Gayheart was born in Hazard, Kentucky and raised in Pine Top, Kentucky, the daughter of Floneva (née Slone), who worked as a Mary Kay independent beauty consultant, and Curtis Gayheart, a miner and coal truck driver. She is of Irish, Italian, and German descent. She moved to New York City at the age of 15 where she completed her education at New York's Professional Children's School and the Lee Strasberg Institute.

In her first film role, Gayheart appeared in Brett Ratner's New York University (NYU) short film Whatever Happened to Mason Reese? (1990) starring Mason Reese. She also appeared in the Ratner-directed music video "Nuttin' But Love" performed by Heavy D and the Boyz.

Gayheart's break into the television industry was a series of television commercials for Noxzema in the early 1990s, earning her the moniker "The Noxzema Girl". The commercials began airing in 1991 and brought her recognition. (This was also referenced at the end of the film Urban Legend, when one of the new college students remarks, "And Brenda [Gayheart's character] was the Noxzema Girl.")

In 1992, Gayheart was cast in her first major role in the soap opera Loving as Hannah Mayberry. In 1993 and 1994, she had a recurring role in the Vanishing Son action pack series. In 1994 and 1995, Gayheart played Bess Martin in the science fiction series Earth 2. In 1995, she had a recurring role as Antonia Marchette in the series Beverly Hills, 90210 as Luke Perry's love interest.

Gayheart has been a regular on the 1999 television series Wasteland, Dead Like Me, Vanished, and had a recurring role on Nip/Tuck. She was initially hired to play the part of Inara Serra in the 2002 series Firefly, but was dropped after only one day of filming. Morena Baccarin took over the role, and none of Rebecca's scenes were used.

In 2007, Gayheart guest-starred on Ugly Betty as Jordan, an ex-girlfriend of Alexis Meade. The following year, Gayheart starred in the Broadway comedy, Boeing-Boeing opposite Christine Baranski, Mark Rylance, Greg Germann, Paige Davis and Missi Pyle.

For a time, Gayheart was engaged to director Brett Ratner[citation needed]. She and actor Eric Dane were married on October 29, 2004. Gayheart and Dane welcomed their first child, a daughter, Billie Beatrice, on March 3, 2010.

Dane told Flaunt Magazine about how they met:

‘Yeah, sure.’ Ten months later, we were married.

On August 17, 2009, a home video was posted on the website Gawker.com that showed former Miss Teen USA contestant Kari Ann Peniche with Dane and wife Rebecca Gayheart. The three are nude and apparently under the influence of alcohol and drugs. They appear to be engaged in some sort of sexual situation, even though the 12 minute video was edited down to just 3 minutes. Midway through the clip, Gayheart says she has to lie down because she's "so high." Kari Ann Peniche also makes a reference to drugs being "fun," and there is a conversation referencing cocaine. The clip ends with both Gayheart and Peniche in a bathtub together, while Dane runs the camera and adds commentary.