Rutherford began her career on daytime soap operas. She appeared on Loving in 1987, and later was cast as a regular on Generations from 1989 to 1991. In 1992 she joined the cast of the ABC drama Homefront.
From 1993 to 1994, Rutherford was a regular on the Fox series The Adventures of Brisco County, Jr. as Dixie Cousins, a saloon singer and con artist who has a brief romantic encounter with Brisco. In later episodes, Dixie becomes Brisco’s primary love interest.
She also appeared in the film I Love Trouble. She also starred in the short-lived series The Great Defender as Frankie Collet (1995), and in Kindred: The Embraced as Caitlin Byrne (1996). In 1996, Rutherford joined the cast of Fox primetime soap opera Melrose Place, as Megan Lewis. She starred on the show until 1999.
She also starred as Christine Hamilton in the horror film Scream 3 (2000). From 2003 to 2004, she starred in the ABC drama Threat Matrix as Special Agent Frankie Ellroy Kilmer. In next television season she starred in another short-lived series, E-Ring, on NBC. In 2007, Rutherford began starring as Lily van der Woodsen in the CW series Gossip Girl. The series ended in 2012. Rutherford has also starred in a number of Lifetime movies. In 2016, Rutherford was cast in the ABC thriller Quantico playing the recurring role of Laura Wyatt. Since 2018, Rutherford has played the recurring role of Melissa Daniels in the reboot series Dynasty.