Nettles began performing in Georgia in her childhood at school assemblies, Georgia 4-H's Clovers & Co., her Southern Baptist church, and regional theater.

While a student at Agnes Scott College, Nettles and Cory Jones (who at the time was studying classical guitar at the University of Georgia) formed Soul Miner’s Daughter. The group, which performed as both an acoustic duo and with a band, released two albums, The Sacred and Profane in 1996 and Hallelujah in 1998, both of which were composed of songs written collaboratively by Jones and Nettles.

In 1999, she formed the Jennifer Nettles Band, with which she produced three studio and two live albums. She and her band were invited to perform at Lilith Fair in 1999 and received the 2000 "Independent Musician of the Year" award from Musician's Atlas.

She moved away from the introspective and sometimes dark themes of her solo work when she teamed up with Kristen Hall and Kristian Bush in 2003 to form Sugarland (Hall has since left the group).

Sugarland was nominated for a Grammy award in the Best New Artist category. They did not win the award, but Nettles and Bush performed on the nationally televised awards show in February 2006, and Nettles presented a Lifetime Achievement Award to Merle Haggard and presented the award for Best Country Group.

Nettles also is seeing success outside of Sugarland. A duet performance with rock band Bon Jovi, "Who Says You Can't Go Home?", has reached No. 23 on the Billboard Hot 100 chart and No. 1 on the Billboard Hot Country Singles chart. The video for the song won a CMT Music Award in 2006 for "Collaborative Video Of The Year". In February of 2007, Nettles and Bon Jovi won a Grammy for Best Country Vocal Collaboration. In 2006, Kristen Hall left the group and Nettles and Bush continued on as a duo releasing "Enjoy the Ride" in November of 2006.

Nettles grew up in the small town of Douglas, Georgia US. She divorced her husband Todd Van Sickle in March 2007. She currently lives in the North Georgia Mountains near Dahlonega, Georgia.