Fournette was listed as questionable for Week 3 after missing last week's win over the Patriots with a hamstring injury.
Sources had indicated on Saturday night that Fournette was expected back against Tennessee, but the Jaguars have decided to play it safe with the second-year player.
Backup running back T.J. Yeldon was also listed as questionable because of an ankle injury, but he was active.
"We've been playing a lot of tough games," coach Doug Marrone said Friday. "Players are doing the best job they can to get themselves ready to go, but we played a home opener in New York. We played a home opener here. Now we've got a divisional opponent.
"When you look at it, a lot of teams are going through the same thing, just trying to keep everybody as healthy as they possibly can."