He faked out the Giants on an end around play in the third quarter for a 41-yard run, his longest of his career.
The Jaguars' defense was vital, especially in the fourth quarter when the Giants threatened late in the fourth quarter but were stopped on a critical fourth down play when Eli Manning was forced to throw an interception from the Jaguars 36.
Jaguars linebacker Donald Payne recovered a muffed punt in the final minute to secure the victory.
The Jaguars held the Giants to 324 total yards offense, 229 in the second half.
Entering Sunday's season opener, no one knew what to expect out of the 2018 Giants, with their new head coach, Pat Shurmur, their shiny-new-toy running back and the return of a healthy Odell Beckham Jr.
Bortles completed 18-of-33 passes for 176 yards and a touchdown.
The Jaguars also overcame the first-half loss of running back Leonard Fournette.
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Bortles took advantage of play-action passes and led the Jaguars to a scoring drive on their opening possession when Josh Lambo made a 39-yard field goal. The Jaguars led 13-3 at that point and 13-6 at halftime.
Barkley showed that ability on one big run Sunday. The Jaguars finished with 137 yards rushing.
The Giants cut the Jacksonville lead to 13-6 at the half on a 31-yard Rosas field goal with four seconds remaining. Barkley rushed for 106 yards on 18 carries in his first National Football League start after being the No. 2 overall selection in last April's '18 draft.
Beckham had 11 catches for 111 yards.
The Jaguars finished with two sacks: one by defensive end Calais Campbell, one by defensive end Lerentee McCray. The Jaguars stuffed the Giants' first series, but Giants cornerback Janoris Jenkins intercepted Bortles at the Giants 37 with 5:40 remaining in the first quarter. Rosas' 27-yard field goal cut the Jaguars' lead to 13-6 with:04 remaining in the half. The quarter ended with the Jaguars holding that lead.
It's hard to pick up 68-yard runs in the NFL.
Notable: When Lambo converted a 39-yard first-quarter field goal, it marked his 12th consecutive field goal and his 20th in 21 attempts with the team.