He was born in Michle (now part of Prague). His father, a gardener by occupation, was an amateur violinist. He taught his two sons the violin and after discovering the talent of Jan, who was aged five at the time, arranged for him to study with Karel Weber and Karel Ondříček. Aged eight he studied at the Prague Conservatory with Otakar Ševčík, of whose technique he became the most famous representative. As a child, he used to practice 10 to 12 hours a day, or "until my fingers started to bleed." After 1898, he toured as a soloist, soon becoming renowned for his great virtuosity and flawless intonation, and his very full and noble tone. He played a Guarneri del Gesù and also two Stradivarius violins: he acquired the 1715 Stradivarius Emperor in 1910.

After great success following his debut in Vienna, and in London (where he first appeared at a Hans Richter concert in 1900), Kubelík toured in the USA in 1901 for the first time. He made his first appearance for the Royal Philharmonic Society, London, in the season of 1901 - 2, and in 1902 was awarded the Society's Gold Medal (in succession to Eugène Ysaÿe). In 1902 he brought the Czech Philharmonic Orchestra to London, having assisted it financially in the previous year.

In his personal life, in 1903 he married Countess Anna Julie Marie Széll von Bessenyö (* 1 March 1880 in Budapest), with whom he had eight children, five violinist daughters and three sons, among them conductor Rafael Kubelík.

Kubelík made a number of recordings; his acoustic recordings were made for The Gramophone Company, and for Fonotipia / Polydor (who also recorded Váša Příhoda, Franz von Vecsey and Jacques Thibaud). The Gramophone Company recorded him as obbligato to Dame Nellie Melba in 1904, a match which reflected the classical phrasing, tonal purity and security of his art and was an ideal complement to it. Their early version of the Bach - Gounod Ave Maria (G.C. 03033) was recorded twice, in October 1904 and again in February 1905, and this was one of the great early classics of the gramophone, one of those records which 'made' the instrument a popular success, though the double celebrity single - sided title retailed at one guinea. Nine years later (when technology had improved) the partnership was reformed to re-make the record (as 03333), in May 1913 with organ accompaniment and again in October 1913. It was the latter version which then survived in the inter war catalogue in two - sided form. His 1935 Carnegie Hall concert was also recorded and has been reissued.

He wrote music, including six violin concertos, and continued to perform in public until his death, with a pause between the end of World War I and 1920, during which period he composed. In 1920 he resumed his concert career, but with the advent of Jascha Heifetz, his career dwindled somewhat. In 1917, he was elected as an honorary member of Phi Mu Alpha Sinfonia music fraternity by the fraternity's Alpha Chapter at the New England Conservatory in Boston.

Jan Kubelík died in Prague in 1940, aged 60.

In 1903 his portrait was painted by Philip de László. Carl Sandburg mentions Jan Kubelík in his Chicago Poems, 1916. He is adored by the sisters in Sally Benson's collected short stories which later became the film Meet Me In St. Louis (1944). He is also referred to in Robert Ludlum's 2002 novel The Janson Directive.