In early 1988, Walker joined the cast of NBC daytime soap opera Santa Barbara, playing Andrea Bedford. After leaving daytime later that year, she began appearing on primetime television. During 1989-90 television season, she played the female lead in the short-lived NBC crime drama series True Blue. She later guest-starred on Matlock, L.A. Law, and Tales from the Crypt. She starred in the 1991 NBC television movie Perry Mason and the Case of the Fatal Fashion as the devious daughter of Diana Muldaur. In 1993, she starred alongside Billy Campbell in the short-lived ABC comedy-drama, Moon Over Miami.

During the 1990s, Walker had roles in films such as Universal Soldier (1992), Singles (1992), When the Bough Breaks (1994), While You Were Sleeping (1995), Kazaam (1996), and Happy, Texas (1999). From 1996 to 1999, she played the leading role as Doctor Samantha Waters on the NBC crime drama series Profiler, for which she received Saturn Award for Best Actress on Television nomination. She returned to television with role in the HBO drama series Tell Me You Love Me (2007), and later played villainous Agent June Stahl in the FX crime drama, Sons of Anarchy, (2008–2010). Walker also had the leading role in the short-lived Lifetime police drama series The Protector in 2011. During Seasons 4 and 5 (2014 and 2015) of Longmire, she played Dr. Donna Monaghan, a therapist treating veterans who is the title character's love interest.

In 1992, Walker played the female leading role opposite Jean-Claude Van Damme in the science-fiction action film Universal Soldier directed by Roland Emmerich. Later that year, she played supporting role in the Cameron Crowe romantic comedy Singles. Her first star-billed role was in the 1994 thriller When the Bough Breaks. In 1995, she had supporting roles in Sandra Bullock's box-office hit While You Were Sleeping, and Andy García's flop Steal Big Steal Little. The following year, Walker co-starred alongside Shaquille O'Neal in the fantasy comedy film, Kazaam.

In 1996, Walker was cast in the leading role as Doctor Samantha Waters in the NBC crime drama series Profiler, a role for which she received a Saturn Award for Best Actress on Television nomination in 1998, and a Satellite Award for Best Actress – Television Series Drama. In 1996, she was listed as one of People's "40 Most Fascinating People on TV". Walker starred as the lead during the first three seasons, but left the show in the fall of 1999. She was replaced by Jamie Luner as a new profiler during the show's final season. Later in 1999, Walker co-starred opposite Steve Zahn in the comedy film Happy, Texas, and played the lead role in the Lifetime television movie, If You Believe.

On June 14, 1997, Walker married John Landgraf, then a Producer for NBC, at her parents' home in Santa Fe. The couple have three sons.

In 2002, after three years on hiatus, Walker returned to star in and produce the ABC pilot comedy called My Wonderful Life. It never got picked up. In 2005, she released a documentary film For Norman... Wherever You Are as Director and Producer, which screened at several film festivals. She returned to the New York stage, appearing in a number of Off-Broadway productions. From 2005 to 2010, Walker played many guest roles on television, including Sleeper Cell, ER, The Shield, Boston Legal, Law & Order, CSI: Crime Scene Investigation, Southland, and Law & Order: Special Victims Unit.

In 2007, Walker starred in the HBO drama series about sexual relationships, Tell Me You Love Me; unlike the other regular characters on the show, her character refrained from nudity or sex during the course of the season, but in the season finale, Walker appeared in a graphic nude masturbation scene. The series was cancelled after a single season. Later that year, she played the lead role in, and was a scriptwriter for, a thriller. In 2009, she co-starred in films Toe to Toe and Wonderful World opposite Matthew Broderick. From 2008 to 2010, Walker had the recurring role as ATF Agent June Stahl, a major antagonist on the FX crime drama series Sons of Anarchy.

In 2011, Walker played the leading role in the Lifetime police drama series The Protector, which revolves around a single mother who struggles to balance her family and professional life as an LAPD homicide detective. The series was canceled after one season. In 2014, she played Chyler Leigh' lead character's mother in the short-lived NBC police procedural, Taxi Brooklyn. She made her feature scripted film directing and writing debut with Sex, Death and Bowling starring Selma Blair. In 2015, she was cast in the recurring roles in the Netflix crime drama Longmire, and USA Network thriller, Colony.