She is a 1992 graduate of Lubbock High School. Natalie is the daughter of country musician Lloyd Maines. She is married to actor Adrian Pasdar; they have two children, Jackson Slade Pasdar (born 2001) and Beckett Finn Pasdar (born 2004).

Natalie joined the all-female country music group The Dixie Chicks in 1995. The group has been performing since 1989.

Natalie is the lead singer in the group and also plays acoustic guitar in concert.

In March of 2003, Natalie Maines provoked controversy and anger from country music fans by saying, during a concert in London, that she was "ashamed" that President George W. Bush was from her home state of Texas. Following the uproar and some calls for a boycott, the singer issued an apology: "As a concerned American citizen, I apologize to President Bush because my remark was disrespectful. I feel that whoever holds that office should be treated with the utmost respect. We are currently in Europe and witnessing a huge anti-American sentiment as a result of the perceived rush to war. While war may remain a viable option, as a mother, I just want to see every possible alternative exhausted before children and American soldiers' lives are lost. I love my country. I am a proud American."

Some of her fans remained supportive, while others were disappointed in her political stance.