Metcalf attended Illinois State University and earned her Bachelor of Arts in Theater in 1976. While at ISU, she met fellow theater students, among them John Malkovich, Glenne Headly, Joan Allen, Terry Kinney, and Jeff Perry, the latter two of whom, along with Perry’s high school classmate Gary Sinise, went on to establish Chicago’s famed Steppenwolf Theatre Company. Metcalf began her professional career at Steppenwolf, of which she was a charter member. Metcalf went to New York to appear in an Off-Broadway Steppenwolf production of Balm in Gilead at Circle Repertory in 1984 for which she received the 1984 Obie Award for Best Actress and a 1984–85 Theatre World Award (for best debut in a Broadway or Off-Broadway performance). Metcalf was praised for her performance as Darlene, and was specifically singled out for her 20-minute act two monologue. Chicago critic Richard Christiansen stated:

Metcalf has appeared in several television series, including being a cast member for a single episode of Saturday Night Live—the final episode of the show’s tumultuous 1980–1981 season. In 1981, she appeared as a feature player on the first Dick Ebersol-produced episode of Saturday Night Live following the firing of Jean Doumanian. She appeared in a Weekend Update segment about taking a bullet for the president of the United States. Because of the sketch show’s severe decline in quality at the time and the 1981 Writers Guild of America strike, the show was put on hiatus for retooling. Metcalf was not asked back to be a cast member.

In 1983, Metcalf married Jeff Perry, co-founding member of Steppenwolf Theatre Company. They had a daughter, Zoe Perry, in 1983. They divorced in 1986.

Metcalf was born in Carbondale, Illinois, the eldest of three children. She, her brother James and her sister Linda were raised in Edwardsville, Illinois, which she has said “isn’t anywhere near a theatre.” Her father, James, was the budget director at Southern Illinois University-Edwardsville at the time of his sudden death in 1984. Her mother, Libby, was a librarian. Her great-aunt was the Pulitzer Prize-winning playwright Zoë Akins. She is an alumna of Illinois State University, class of 1976.

Metcalf later began a relationship with Matt Roth, the Roseanne co-star who played her abusive boyfriend Fisher. By November 1993, they had a son, Will, and eventually married. They also worked together on occasion, including in the 1994 feature film thriller Blink and the 1998 drama Chicago Cab; they also appeared together in an episode of Desperate Housewives. Their daughter, Mae Akins, was born in 2005 via surrogate. They had a second son, Donovan, whom they fostered at six years old in 2006 and permanently adopted. On November 26, 2008, Metcalf and Roth separated. In September 2011, Roth filed for divorce, citing irreconcilable differences. In May 2014, the divorce was finalized.

She subsequently appeared with Norm Macdonald on The Norm Show (or Norm), which ran for three seasons (1999–2001), and was also a regular character on the 2003 Nathan Lane series Charlie Lawrence, which was cancelled after the airing of two episodes. Metcalf has made guest appearances on Absolutely Fabulous, Malcolm in the Middle, My Boys, Dharma & Greg, Frasier, Without a Trace, 3rd Rock from the Sun, The Big Bang Theory, and Monk. She has been nominated for the Emmy Award as Outstanding Guest Actress In A Comedy Series for the latter three listed roles. In The Big Bang Theory she played Sheldon Cooper’s mother. In the prequel spinoff Young Sheldon her daughter Zoe Perry has been cast as the same character. In December 2018, Metcalf was nominated for a Critics’ Choice Award for the best supporting actress in a comedy series for The Conners, competing against her own daughter Zoe Perry who was nominated in the same category for her role in Young Sheldon. Neither won.

Metcalf relocated to New York City and began to work in both film and theater, including such productions as David Mamet’s November on Broadway in 2008.

Metcalf took a recurring role on Desperate Housewives, for which she received a Primetime Emmy Award nomination for Best Guest Actress in a Comedy Series, and appeared alongside her ex-husband Jeff Perry in an episode of Grey’s Anatomy. In Fall 2008, Metcalf starred in the CW dramedy Easy Money, as the matriarch of a family of loan sharks. The series was canceled after three episodes. Alongside this role, she was the lead actress in the short-lived CBS sitcom The McCarthys (2014–15).

In June 2009, Metcalf starred in Justin Tanner’s play, Voice Lessons with French Stewart, in Hollywood before beginning rehearsals to play Kate Jerome in the Broadway revival of Neil Simon’s semi-autobiographical plays Brighton Beach Memoirs and Broadway Bound, directed by David Cromer. The former production’s run, however, lasted for nine performances in October 2009, and the latter was canceled before opening.

Voice Lessons, however, with its original cast intact, went on to three more runs — one Off-Broadway in May 2010, another in Hollywood in May 2011, and another in Chicago in May 2016.

In September 2010, Metcalf returned to Steppenwolf and starred in Lisa D’Amour’s play Detroit.

In 2011, she appeared in the Off-Broadway play The Other Place by Sharr White, directed by Joe Mantello. She won the 2011 Lucille Lortel Award, Outstanding Lead Actress, and the 2011 Obie Award, for her performance.

In 2012, Metcalf joined David Suchet in a West End production of Eugene O’Neill’s Long Day’s Journey into Night, for which she was nominated for the Evening Standard Theatre Award for Best Actress.

In 2013, The Other Place transferred to Broadway with Metcalf reprising her role and earning Tony and Drama League nominations. She starred with her real-life daughter, Zoe Perry.

In 2013, Metcalf starred in Bruce Norris’s Off-Broadway play Domesticated with Jeff Goldblum at the Mitzi Newhouse Theater of Lincoln Center. She was nominated for the 2014 Drama League Award, Distinguished Performance and the 2014 Drama Desk Award for Outstanding Actress in a Play for her performance.

From 2013 to 2015, Metcalf starred in HBO comedy series Getting On. Metcalf was nominated for the Primetime Emmy Award for Best Leading Actress in a Comedy Series for the final season.

In 2015, she took the role of Annie Wilkes in the Broadway production of Stephen King’s Misery, opposite Bruce Willis. The play premiered on November 15, 2015 at the Broadhurst Theatre. It received mixed reviews from critics, however Metcalf’s performance was widely acclaimed. She was nominated for the Tony Award for Best Actress in a Play, her third Tony nomination overall.

In 2016, Metcalf took a role in the third episode of Louis C.K.’s self-funded show Horace and Pete. She was nominated for the Primetime Emmy Award for Guest Actress in a Drama for her performance.

Metcalf appeared on Broadway in Lucas Hnath’s A Doll’s House, Part 2 with Chris Cooper at the John Golden Theatre. which opened in April 2017. She received critical acclaim for her performance and earned her first Tony Award, winning for Best Actress in a Play.

Metcalf is perhaps best known for her role as Jackie Harris, the multiple-careered, low self-esteemed, amiable sister of the title character, in the hit series Roseanne. Her performance garnered three consecutive Emmy Awards. Roseanne originally ran from 1988 to 1997. On April 28, 2017, it was announced that a revival of Roseanne was in the works and that Metcalf along with most of the original cast and some of the producers would return for the limited series that was being shopped around with ABC and Netflix the frontrunners to land the show. On May 16, 2017 producers confirmed that eight episodes would air mid-season in 2018 on ABC. On May 29, 2018, in the wake of racist remarks by Barr posted on Twitter regarding Valerie Jarrett (an advisor of former president Barack Obama), ABC cancelled the revival after a single season. Metcalf appeared as Jackie over the show’s entire run. Metcalf reprised her role in The Conners, a spinoff of Roseanne without Barr’s involvement which premiered in fall 2018. In 2018, Metcalf’s performance on the revival of Roseanne was nominated for the Primetime Emmy Award for Best Comedy Actress in a Supporting Role.

In 2017, Metcalf received universal critical acclaim for her performance in Greta Gerwig’s coming of age film Lady Bird starring alongside Saoirse Ronan and Tracy Letts. For her performance, she was nominated for numerous awards, including the Academy Award, Golden Globe, SAG, BAFTA, Critics Choice and the Independent Spirit Award.

In 2018, Metcalf performed in the Broadway revival of Three Tall Women with Glenda Jackson and Allison Pill at the John Golden Theatre. She won her second consecutive Tony Award, this time for Best Featured Actress in a Play.