[4] Barber graduated from Cave Spring High School of Roanoke, Virginia in 1993, and was a standout in football, wrestling, and track.

On January 3, 1997, Barber announced his decision to forgo his final season of eligibility and enter the 1997 NFL draft.

Ronde and Tiki Barber became only the fourth set of twins to be selected in the same draft in NFL history, following Rich and Ron Saul (1970), Reggie and Raleigh McKenzie (1985), and Keith and Kerry Cash (1991).

Improving from his previous seasons, Barber posted 74 tackles, two interceptions (60 yards), one sack, and 16 passes deflected.

[20][21] In 2001, Barber started 16 of 16 regular season games and made 71 tackles, 1 sack, 14 passes deflected, and 10 interceptions (86 yards).

[17] Barber returned an intercepted pass from Aaron Brooks 36 yards for a touchdown in the Week 15 (December 23) win over the New Orleans Saints.

[15] Starting all 16 games again this season, Barber made 79 tackles, 3 sacks, 17 passes deflected, and returned 2 interceptions for 9 yards.

[15] In the NFC Championship game on January 19, 2003, Barber picked off a pass from Philadelphia Eagles quarterback Donovan McNabb and returned the interception 92 yards for a touchdown late in the fourth quarter to seal Tampa's 27–10 road victory and brought Veterans Stadium to near-silence.

[23][24][25] Bob Ford of The Philadelphia Inquirer labeled this game as "the most ignominious defeat of Andy Reid's head coaching career" because of Barber's interception return.

[26] In 2011, Tampa Bay Times sportswriter Rick Stroud ranked Barber's interception return for touchdown in the NFC Championship game as the greatest scoring play in team history.

[27] In Tampa's 48–21 victory over the Oakland Raiders in Super Bowl XXXVII on January 26, 2003, Barber made five tackles.

[17] In Week 5 (October 6), a 38–35 overtime loss to the Indianapolis Colts, Barber returned an intercepted pass from Peyton Manning 29 yards for a touchdown.

[28] Starting all 16 regular-season games, Barber made 98 tackles, 3 sacks, 10 passes defended, 3 interceptions returned for 23 yards, and 2 forced fumbles.

[29] On December 11, in the Week 14 game against the Carolina Panthers, he became the first cornerback in the history of the NFL to record at least 20 interceptions and 20 sacks in his career.

[17] On October 22 (Week 7) against Philadelphia, Barber became the first player in Buccaneers history to return two interceptions for touchdowns in one regular-season game.

[15] In 16 games, Barber made 58 tackles, one sack, 14 passes deflected, 2 interceptions returned for 32 yards (including a 29-yard touchdown run), and one forced fumble.

[17] On November 25, 2007, he took the record from Donnie Abraham for the most interceptions in Buccaneers team history by catching his 32nd against the Washington Redskins.

[33] In a 37–3 victory over the Atlanta Falcons on Week 15 (December 16), Barber returned an interception of Chris Redman 29 yards for a touchdown.

[36] In the 2009 regular season, Barber made 77 tackles, 2 sacks, 6 passes defended, and 2 forced fumbles in 16 games (all starts).

[37] In the Week 9 win over the Green Bay Packers, Barber returned a punt blocked by Geno Hayes 31 yards for a tying touchdown.

[38] Along with Tony Dungy, Derrick Brooks, and Warren Sapp, Barber was voted to the NFL 2000s All-Decade Team prior to the 2010 Pro Bowl.

[41] On November 21, 2010, Barber became the only player in NFL history with at least 25 sacks (26 total) and 40 interceptions with a 21–0 win over the San Francisco 49ers.

[17][43] On October 23 (Week 7), in an NFL International Series game against the Chicago Bears held in London, England, Barber recorded his first career safety when he tackled Matt Forte in the end zone.

[45] Barber started all 16 games for the 13th straight season and had 91 tackles, 13 passes defended, 4 interceptions returned for 160 yards, and a forced fumble.