Muybridge was born Edward James Muggeridge at Kingston upon Thames, London, England. He is believed to have changed his first name to match that of King Eadweard as shown on the plinth of the Kingston coronation stone, which was re-erected in Kingston in 1850. Although he did not change his first name until the 1870s, he changed his surname to Muygridge early in his San Francisco career and then changed it again to Muybridge at the launch of his photographic career or during the years between.

In 1855 Muybridge arrived in San Francisco, starting his career as a publisher's agent and bookseller. He left San Francisco at the end of that decade, and after a stagecoach accident in which he received severe head injuries returned to England for a few years.

He reappeared in San Francisco in 1866 as a photographer named Muybridge and rapidly became successful in the profession, focusing principally on landscape and architectural subjects, although his business cards also advertised his services for portraiture. His photographs were sold by various photographic entrepreneurs on Montgomery Street (most notably the firm of Bradley & Rulofson), San Francisco's main commercial street, during those years.

Muybridge began to build his reputation in 1867 with photos of Yosemite and San Francisco (many of the Yosemite photographs reproduced the same scenes taken by Carleton Watkins). Muybridge quickly gained notice for his landscape photographs, which showed the grandeur and expansiveness of the West. The images were published under the pseudonym “Helios.” In the summer of 1868 Muybridge was commissioned to photograph one of the U.S. Army's expeditions.

In 1872, former Governor of California Leland Stanford, a businessman and racehorse owner, had taken a position on a popularly debated question of the day: whether all four of a horse's hooves left the ground at the same time during a gallop. Stanford sided with this assertion, called "unsupported transit", and took it upon himself to prove it scientifically. Stanford sought out Muybridge and hired him to settle the question. In 1877, Muybridge settled Stanford's question with a single photographic negative showing Stanford's racehorse Occident airborne in the midst of a gallop. This negative was lost, but it survives through woodcuts made at the time. By 1878, spurred on by Stanford to expand the experiment, Muybridge had successfully photographed a horse in fast motion. This series of photos taken in Palo Alto, California, is called Sallie Gardner at a Gallop or The Horse in Motion, and shows that the hooves do all leave the ground — although not with the legs fully extended forward and back, as contemporary illustrators tended to imagine, but rather at the moment when all the hooves are tucked under the horse as it switches from "pulling" with the front legs to "pushing" with the back legs.

Eventually, Muybridge and Stanford had a major falling-out concerning this research on equine locomotion: Stanford published a book The Horse in Motion which gave no credit to Muybridge despite containing his photos and his research, possibly because Muybridge lacked an established reputation in the scientific community. As a result of Muybridge's lack of credit for the work, the Royal Society withdrew an offer to fund his stop-motion photography. Muybridge subsequently filed a lawsuit against Stanford, and lost.

In 1874, still living in the San Francisco Bay Area, Muybridge discovered that his wife had a lover, a Major Harry Larkyns. On 17 October 1874, he sought out Larkyns; said, "Good evening, Major, my name is Muybridge and here is the answer to the letter you sent my wife"; he then killed the Major with a gunshot. Muybridge believed Larkyns to be his son's true father, although as an adult, the son bore a remarkable resemblance to Muybridge. Muybridge was put on trial for murder, but was acquitted as a "justifiable homicide". The inquiry interrupted his horse photography experiment, but not his relationship with Stanford, who paid for his criminal defense. An interesting aspect of Muybridge's defense was a plea of insanity due to a head injury Muybridge sustained following his stagecoach accident. Friends testified that the accident dramatically changed Muybridge's personality from genial and pleasant to unstable and erratic. The jury dismissed the insanity plea.

After the acquittal, Muybridge left the United States for a time to take photographs in Central America, returning in 1877. He had his son, Florado Helios Muybridge (nicknamed "Floddie" by friends), put in an orphanage. As an adult, Floddie worked as a ranch hand and gardener. In 1944 he was hit by a car in Sacramento and killed.

This episode in Muybridge's life is the subject of The Photographer, a 1982 opera by Philip Glass, with words drawn from the trial and Muybridge's letters to his wife.

At the Chicago 1893 World's Columbian Exposition, Muybridge gave a series of lectures on the Science of Animal Locomotion in the Zoopraxographical Hall, built specially for that purpose in the "Midway Plaisance" arm of the exposition. He used his zoopraxiscope to show his moving pictures to a paying public making the Hall the very first commercial movie theater.

At the University of Pennsylvania and the local zoo Muybridge used banks of cameras to photograph people and animals to study their movement. The models, either entirely nude or with very little clothing, were photographed in a variety of undertakings, ranging from boxing, to walking down stairs, to throwing water over one another and carrying buckets of water. Between 1883 and 1886 he made a total of 100,000 images, working under the auspices of the University of Pennsylvania. They were published as 781 plates comprising 20,000 of the photographs in a collection titled Animal Locomotion. Muybridge's work stands near the beginning of the science of biomechanics and the mechanics of athletics.

Recent scholarship has pointed to the influence of Étienne Jules de Marey on Muybridge's later work. Muybridge visited Marey's studio in France and saw Marey's stop-motion studies before returning to the U.S. to further his own work in the same area. However, whereas Marey's scientific achievements in the realms of cardiology and aerodynamics (as well as pioneering work in photography and chronophotography) are indisputable, Muybridge's efforts were to some degree artistic rather than scientific. As Muybridge himself explained, in some of his published sequences he substituted images where exposures failed, in order to illustrate a representative movement (rather than producing a strictly scientific recording of a particular sequence).

Similar setups of carefully timed multiple cameras are used in modern special effects photography with the opposite goal of capturing changing camera angles with little or no movement of the subject. This is often dubbed "bullet time" photography.

Eadweard Muybridge returned to his native England in 1894, published two further, popular books of his work, and died on 8 May 1904 in Kingston upon Thames while living at the home of his cousin Catherine Smith, Park View, 2 Liverpool Road. The house has a British Film Institute commemorative plaque on the outside wall. Muybridge was cremated and his ashes interred at Woking.

Many of his photographic sequences have been published since the 1950s as artists' reference books. In 1985 the music video for Larry Gowan's single "(You're A) Strange Animal" prominently featured animation rotoscoped from Muybridge's work. In 1986 the galloping horse sequence was used in the background of the John Farnham music video for the song Pressure Down. In 1993, U2 made a video to their song "Lemon" into a tribute to Muybridge's techniques. In 2004, the electronic music group The Crystal Method made a music video to their song "Born Too Slow" which was based on Muybridge's work, including a man walking in front of a background grid.

A documentary of his life and work, titled Eadweard Muybridge, Zoopraxographer was made by filmmaker Thom Andersen, in 1974.
Composer Philip Glass's 1982 opera The Photographer is based on Muybridge's murder trial, the libretto including text from the transcript. A promotional music video of an excerpt of the opera dramatized the murder and trial and included a considerable number of Muybridge images. Kingston University, London, UK has a building named in recognition of his work as one of Britain's most influential photographers. In 2007, Canadian poet Rob Winger wrote Muybridge's Horse: a poem in three phases, a long poem nominated for the Governor General's Award for Literature, Trillium Book Award for Poetry, and Ottawa Book Award. It documented his life and obsessions in a 'poetic-photographic' style. It won the CBC Literary Award for Poetry.