Anaïs Nin was born in Neuilly, France, to two artistic parents. Her father, Joaquín Nin, was a Spanish pianist and composer, living in Cuba when he met her mother Rosa Culmell, who was a classically trained singer in Cuba of French and Danish descent. Her father's grandfather had fled France during the Revolution, going first to Saint - Domingue, then New Orleans, and finally to Cuba where he helped build that country's first railway.

Nin was raised a Roman Catholic and spent her childhood and early life in Spain. After her parents separated, her mother moved Anaïs and her two brothers, Thorvald Nin and Joaquin Nin - Culmell, from Barcelona to New York City. According to her diaries, Volume One, 1931 – 1934, Nin abandoned formal schooling at the age of sixteen years and later began working as a dancer and model. After being in America for several years, Nin had forgotten how to speak Spanish, but retained her French and became fluent in English.

On March 3, 1923, in Havana, Cuba, Nin married her first husband, Hugh Parker Guiler (1898 – 1985), a banker and artist, later known as "Ian Hugo" when he became a maker of experimental films in the late 1940s. The couple moved to Paris the following year, where Guiler pursued his banking career and Nin began to pursue her interest in writing; in her diaries she also mentions having trained as a flamenco dancer in Paris in the mid-to-late 1920s. Her first published work was a critical evaluation of D.H. Lawrence called D.H. Lawrence: An Unprofessional Study, which she wrote in sixteen days. She also explored the field of psychotherapy, studying under the likes of Otto Rank, a disciple of Sigmund Freud. Nin was briefly a patient of Carl Jung.

Nin left Paris in the late summer of 1939, when residents from overseas were urged to leave France due to the upcoming war and returned to New York City with Guiler (who was, on his own wish, all but edited out of her diaries published in her lifetime and whose role in her life is therefore difficult to gauge). During the war, Nin sent her books to Frances Steloff of the Gotham Book Mart in New York for safekeeping.

According to her diaries,Vol.1, 1931 – 1934, Nin shared a bohemian lifestyle with Henry Miller during her time in Paris. Her husband Guiler is not mentioned anywhere in the published edition of the 1930s parts of her diary (Vol.1 – 2) although the opening of Vol.1 makes it clear that she is married, and the introduction suggests her husband refused to be included in the published diaries. Nin appeared in the Kenneth Anger film Inauguration of the Pleasure Dome (1954) as Astarte; in the Maya Deren film Ritual in Transfigured Time (1946); and in Bells of Atlantis (1952), a film directed by Guiler under the name "Ian Hugo" with a soundtrack of electronic music by Louis and Bebe Barron. The diaries edited by her second husband, after her death, tell that her union with Henry Miller was very passionate and physical, and that she believed that it was his fetus that she aborted.

In 1947, at the age of 44, she met former actor Rupert Pole in a Manhattan elevator on her way to a party. The two ended up dating and moved in together; Pole was sixteen years her junior. On March 17, 1955, she married him at Quartzsite, Arizona, returning with Pole to live in California. Guiler remained in New York City and was unaware of Nin's second marriage until after her death in 1977, though biographer Deirdre Bair alleges that Guiler knew what was happening while Nin was in California, but consciously "chose not to know". Nin referred to her simultaneous marriages as her "bicoastal trapeze". According to Deidre Bair:

In 1966, Nin had her marriage with Pole annulled, due to the legal issues arising from both Guiler and Pole having to claim her as a dependent on their federal tax returns. Though the marriage was annulled, Nin and Pole continued to live together as if they were married, up until her death in 1977.

After Guiler's death in 1985, the unexpurgated versions of her journals were commissioned by Pole. Pole died in July 2006.

Nin often cited authors Djuna Barnes and D.H. Lawrence as inspirations. She states in Volume One of her diaries that she drew inspiration from Marcel Proust, André Gide, Jean Cocteau, Paul Valéry, and Arthur Rimbaud.

Nin once worked at Lawrence R. Maxwell Books located at 45 Christopher Street.

Anaïs Nin is perhaps best remembered as a diarist. Her journals, which span several decades, provide a deeply explorative insight into her personal life and relationships. Nin was acquainted, often quite intimately, with a number of prominent authors, artists, psychoanalysts, and other figures, and wrote of them often, especially Otto Rank. Moreover, as a female author describing a primarily masculine constellation of celebrities, Nin's journals have acquired importance as a counter balancing perspective.

Previously unpublished works are coming to light in A Café in Space, the Anais Nin Literary Journal, which most recently includes "Anais Nin and Joaquín Nin y Castellanos: Prelude to a Symphony — Letters between a father and daughter."

Nin is hailed by many critics as one of the finest writers of female erotica. She was one of the first women to explore fully the realm of erotic writing, and certainly the first prominent woman in the modern West to write erotica. Before her, erotica written by women was rare, with a few notable exceptions, such as the work of Kate Chopin.

According to Volume I of her diaries, 1931 – 1934, published in 1966 (Stuhlmann), Nin first came across erotica when she returned to Paris with her [husband,] mother and two brothers in her late teens. They rented the apartment of an American man who was away for the summer, and Nin came across a number of French paperbacks: "One by one, I read these books, which were completely new to me. I had never read erotic literature in America… They overwhelmed me. I was innocent before I read them, but by the time I had read them all, there was nothing I did not know about sexual exploits… I had my degree in erotic lore."

Faced with a desperate need for money, Nin, Miller and some of their friends began in the 1940s to write erotic and pornographic narratives for an anonymous "collector" for a dollar a page, somewhat as a joke. (It is not clear whether Miller actually wrote these stories or merely allowed his name to be used.) Nin considered the characters in her erotica to be extreme caricatures and never intended the work to be published, but changed her mind in the early 1970s and allowed them to be published as Delta of Venus and Little Birds.

Nin was a friend, and in some cases lover, of many leading literary figures, including Henry Miller, Antonin Artaud, Edmund Wilson, Gore Vidal, James Agee, James Leo Herlihy, and Lawrence Durrell. Her passionate love affair and friendship with Miller strongly influenced her both as a woman and an author. The rumor that Nin was bisexual was given added circulation by the Philip Kaufman film Henry & June. This rumor is dashed by at least two encounters Nin writes about in her third unexpurgated journal, Fire. The first is with a patient of Nin's (Nin was working as a psychoanalyst in New York at the time), Thurema Sokol, with whom nothing physical occurs. She also describes a ménage à trois in a hotel, and while Nin is attracted to the other woman, she does not respond completely. Nin confirms that she is not bisexual in her unpublished 1940 diary when she states that although she could be attracted erotically to some women, the sexual act itself made her uncomfortable. What is irrefutable is her sexual attraction to men.

Nin's first unexpurgated journal, Henry and June, makes it clear, despite the notion to the contrary, that she did not have sexual relations with Miller's wife, June. While Nin was stirred by June to the point where she says (paraphrasing), "I have become June," she did not consummate her erotic feelings for her. Still, to both Anais and Henry, June was a femme fatale — irresistible, cunning, erotic. Nin gave June money, jewelry, clothes, often times leaving herself broke. In her second unexpurgated journal, Incest, she wrote that she had an incestuous relationship with her father, which was graphically described. When Nin's father learned of the title of her first book of fiction, House of Incest, he feared that the true nature of their relationship would be revealed, when, in fact, it was heavily veiled in Nin's text.

The explosion of the feminist movement in the 1960s gave feminist perspectives on Nin's writings of the past twenty years, which made Nin a popular lecturer at various universities; contrarily, Nin disassociated herself from the political activism of the movement.

In 1973 Anaïs Nin received an honorary doctorate from the Philadelphia College of Art. She was elected to the United States National Institute of Arts and Letters in 1974. She died in Los Angeles, California on January 14, 1977 after a three year battle with cancer. Her body was cremated, and her ashes were scattered over Santa Monica Bay in Mermaid Cove. Her first husband, Hugh Guiler, died in 1985, and his ashes were scattered in the cove as well. Rupert Pole was named Nin's literary executor, and he arranged to have new unexpurgated editions of Nin's books and diaries published between 1985 and his death in 2006.

Philip Kaufman directed the 1990 film Henry & June based on Nin's novel Henry and June: From the Unexpurgated Diary of Anaïs Nin. She was portrayed in the film by Maria de Medeiros.