Jerome was inspired by his older sister Blandina's love for the theatre, and he decided to try his hand at acting in 1877, under the stage name Harold Crichton. He joined a repertory troupe that produced plays on a shoestring budget, often drawing on the actors' own meagre resources – Jerome was penniless at the time – to purchase costumes and props. After three years on the road with no evident success, the 21-year-old Jerome decided that he had enough of stage life and sought other occupations. He tried to become a Journalist, writing essays, satires, and short stories, but most of these were rejected. Over the next few years, he was a school Teacher, a packer, and a solicitor's clerk. Finally, in 1885, he had some success with On the Stage – and Off (1885), a comic memoir of his experiences with the acting troupe, followed by Idle Thoughts of an Idle Fellow (1886), a collection of humorous essays which had previously appeared in the newly founded magazine, Home Chimes, the same magazine that would later serialise Three Men in a Boat.

Other works include the essay collections Idle Thoughts of an Idle Fellow (1886) and Second Thoughts of an Idle Fellow; Three Men on the Bummel, a sequel to Three Men in a Boat, and several other novels.

On 21 June 1888, Jerome married Georgina Elizabeth Henrietta Stanley Marris ("Ettie"), nine days after she divorced her first husband. She had a daughter from her previous, five-year marriage nicknamed Elsie (her actual name was also Georgina). The honeymoon took place on the Thames "in a little boat," a fact that was to have a significant influence on his next and most important work, Three Men in a Boat.

Jerome sat down to write Three Men in a Boat as soon as the couple returned from their honeymoon. In the novel, his wife was replaced by his longtime friends George Wingrave (George) and Carl Hentschel (Harris). This allowed him to create comic (and non-sentimental) situations which were nonetheless intertwined with the history of the Thames region. The book, published in 1889, became an instant success and has never been out of print. Its popularity was such that the number of registered Thames boats went up fifty percent in the year following its publication, and it contributed significantly to the Thames becoming a tourist attraction. In its first twenty years alone, the book sold over a million copies worldwide. It has been adapted to films, TV and radio shows, stage plays, and a musical. Its writing style influenced many humorists and satirists in England and elsewhere.

With the financial security that the sales of the book provided, Jerome was able to dedicate all of his time to writing. He wrote a number of plays, essays, and novels, but was never able to recapture the success of Three Men in a Boat. In 1892, he was chosen by Robert Barr to edit The Idler (over Rudyard Kipling). The magazine was an illustrated satirical monthly catering to gentlemen (who, following the theme of the publication, appreciated idleness). In 1893, he founded To-Day, but had to withdraw from both publications because of financial difficulties and a libel suit.

In 1898, a short stay in Germany inspired Three Men on the Bummel, the sequel to Three Men in a Boat, reintroducing the same characters in the setting of a foreign bicycle tour. The book was nonetheless unable to capture the life-force and historic roots of its predecessor, and it enjoyed only a mild success. In 1902, he published the novel Paul Kelver, which is widely regarded as autobiographical. His 1908 play The Passing of the Third Floor Back introduced a more sombre and religious Jerome. The main character was played by one of the leading actors of the time, Johnston Forbes-Robertson, and the play was a tremendous commercial success. It was twice made into film, in 1918 and in 1935. However, the play was condemned by critics – Max Beerbohm described it as "vilely stupid" and as written by a "tenth-rate writer".

In 1926, Jerome published his autobiography, My Life and Times. Shortly afterwards, the Borough of Walsall conferred on him the title Freeman of the Borough. During these last years, Jerome spent more time at his farmhouse Gould's Grove southeast of Ewelme near Wallingford.

Jerome suffered a paralytic stroke and a cerebral haemorrhage in June 1927, on a motoring tour from Devon to London via Cheltenham and Northampton. He lay in Northampton General Hospital for two weeks before dying on 14 June. He was cremated at Golders Green and his ashes buried at St Mary's Church, Ewelme, Oxfordshire. Elsie, Ettie, and his sister Blandina are buried beside him. His gravestone reads "For we are labourers together with God". A small museum dedicated to his life and works was opened in 1984 at his birth home in Walsall, but it closed in 2008, and the contents were returned to Walsall Museum.