Mathis attended Englewood High School in Jacksonville at the same time as Brett Myers of the Chicago White Sox, and was a letterman in football.

At the conclusion of the pre-draft process, Mathis was projected to be a second or third round pick by NFL draft experts and scouts.

You can't paint a more perfect picture than this, coming home and playing in your hometown.”[7] On July 24, 2003, the Jacksonville Jaguars signed Mathis to a four-year, $3.17 million contract.

He made his professional regular season debut and first career start in the Jacksonville Jaguars' season-opener at the Carolina Panthers and made two combined tackles, a pass deflection, and his first career interception off a pass by quarterback Jake Delhomme in their 24–23 loss.

In Week 6, Mathis was converted to cornerback due to injuries to Jason Craft and Kiwaukee Thomas.

He started in all 16 games in 2003 and made a career-high 81 combined tackles (71 solo), nine pass deflections, and two interceptions.

Head coach Jack Del Rio named Mathis and Dewayne Washington the starting cornerbacks to begin the regular season.

[12] He had his most productive year in 2006, collecting eight interceptions (tied for third in the league) and being voted to the Pro Bowl where he started alongside Champ Bailey.

On December 1, 2008, Mathis sustained a knee injury against the Houston Texans and was put on the injured reserve list, ending his season.

[21] [13] Key In 2006, Mathis appeared as a celebrity contestant on Wheel of Fortune during NFL Players Week.