In 1903, when she was nine years old, her family moved to Santa Cruz, California, to seek a warmer climate and better job opportunities. Her childhood home at 208 Lincoln Street still stands. She attended Santa Cruz High School, where she participated in school theatricals.

Pitts made her stage debut in 1914–15 doing school and local community theater in Santa Cruz. Going to Los Angeles in 1916, at the age of twenty-two, she spent many months seeking work as a film extra. Finally she was discovered for substantive roles in films by Screenwriter Frances Marion. Marion cast Pitts as an orphaned slavey (child of work) in the silent film, A Little Princess (1917), starring Pickford.

Pitts' popularity grew following a series of Universal one-reeler comedies and earned her first feature-length lead in King Vidor's Better Times (1919). The following year she married her first husband, Tom Gallery, with whom she was paired in several films, including Heart of Twenty (1920), Bright Eyes (1921), Patsy (1921) and A Daughter of Luxury (1922). In 1924, the Actress, now a reputable comedy farceuse, was given the greatest tragic role of her career in Erich von Stroheim's ​9 ⁄₂-hour epic Greed (1924). The surprise casting initially shocked Hollywood, but showed that Pitts could draw tears with her doleful demeanor as well as laughs. Having been extensively edited prior to release—the final theatrical cut ran just over two hours—the movie failed initially at the box office, but has since been restored to over four hours and is considered one of the greatest films ever made. Based on her performance, von Stroheim labeled Pitts "the greatest dramatic actress". He also featured her in his films The Honeymoon (1928), The Wedding March (1928), War Nurse (1930) and Walking Down Broadway, released as Hello, Sister! (1933).

Pitts was married to actor Thomas Sarsfield Gallery from 1920 to 1933. Gallery became a Los Angeles boxing promoter and later a TV executive. The couple had two children: Ann Gallery (born 1922) and Donald Michael "Sonny" Gallery (né Marvin Carville La Marr), who they adopted and renamed after the 1926 death of his mother (and Pitts' friend) silent film Actress Barbara La Marr. In 1933, she married John Edward "Eddie" Woodall, with whom she remained until her death.

Pitts enjoyed her greatest fame in the early 1930s, often starring in B movies and comedy short films, teamed with Thelma Todd. She played secondary parts in many films. Her stock persona (a fretful, flustered, worried spinster) made her instantly recognizable and was often imitated in cartoons and other films. She starred in a number of Hal Roach short films and features, and co-starred in a series of feature-length comedies with Slim Summerville. Switching between comedy short films and features, by the advent of sound, she was relegated to comedy roles. A bitter disappointment was when she was replaced in the classic war drama All Quiet on the Western Front (1930) by Beryl Mercer after her initial appearance in previews drew unintentional laughs, despite her intense performance. She had viewers rolling in the aisles in Finn and Hattie (1931), The Guardsman (1931), Blondie of the Follies (1932), Sing and Like It (1934) and Ruggles of Red Gap (1935). In 1936 and 1937 she portrayed Hildegarde Withers in two movies, succeeding Edna May Oliver as the spinster sleuth, but they were not well received.

In the 1940s, she found work in radio, trading banter with Bing Crosby, Al Jolson, W.C. Fields, and Rudy Vallee, among others. Pitts' activities on radio included playing Miss Mamie Wayne on the soap opera Big Sister. She appeared several times on the earliest Fibber McGee and Molly show, playing a dizzy dame constantly looking for a husband.

In 1944, Pitts tackled Broadway, making her debut in the mystery, Ramshackle Inn. The play, written expressly for her, did well, and she took the show on the road in later years. Post-war films continued to give Pitts the chance to play comic snoops and flighty relatives in such fare as Life with Father (1947), but in the 1950s she started focusing on television. This culminated in her best known series role, playing second Banana to Gale Storm on CBS's The Gale Storm Show (1956) (also known as Oh, Susannah) in the role of Elvira Nugent ("Nugie"), the shipboard beautician. In 1961, Pitts was cast opposite Earle Hodgins in the episode "Lonesome's Gal" on the ABC sitcom, Guestward, Ho!, set on a dude ranch in New Mexico. In 1962, Pitts appeared in an episode of CBS's Perry Mason, "The Case of the Absent Artist". Her final role was as Gertie, the switchboard operator in the Stanley Kramer comedy epic It's a Mad, Mad, Mad, Mad World (1963).

ZaSu Pitts was inducted to the Hollywood Walk of Fame on February 8, 1960 for her contribution to motion pictures. Her star is on the south side of the 6500 block of Hollywood Boulevard.

Declining health dominated Pitts' later years, particularly after she was diagnosed with cancer in the mid-1950s. She continued to work, appearing on TV and making brief appearances in The Thrill of It All (1963) and It's a Mad, Mad, Mad, Mad World. She died June 7, 1963, aged 69, in Hollywood and was interred at Holy Cross Cemetery, Culver City. Pitts wrote a book of candy recipes, Candy Hits by ZaSu Pitts, which was published posthumously in 1963.

Eliza Susan Pitts was born in Parsons, Kansas, to Rulandus and Nelly (née Shay) Pitts; she was the third of four children. Her father, who had lost a leg while serving in the 76th New York Infantry in the Civil War, had settled the family in Kansas by the time ZaSu was born.

In 1994, she was honored with her image on a United States postage stamp along with luminaries such as Rudolph Valentino, Clara Bow and Charlie Chaplin as part of The Silent Screen Stars stamp set, designed by caricaturist Al Hirschfeld. In Parsons, Kansas, there is a star tile at the entrance to the Parsons Theatre to commemorate her.