The league's investigation was first reported Friday by BuzzFeed News, which viewed a letter it said was sent from NFL special counsel for investigations Lisa Friel to the Uber driver.
NFL spokesman Brian McCarthy confirmed to ESPN that the "allegation was shared with the NFL and we have reached out to Uber to request any information they may have."
In the letter to the unnamed woman, Friel wrote: "The League has been informed that you may have been the victim of such a violation perpetrated by Tampa Bay Buccaneers player Jameis Winston The league takes allegations of this nature very seriously and has opened an investigation into this matter."
The Buccaneers released a statement that they are aware of an allegation.
"We are in the process of obtaining further information regarding today's media report. We take these matters seriously and are fully supportive of the investigation that is being conducted by the NFL," the team statement said.
Bucs coach Dirk Koetter told reporters Friday he was "just finding out" about the allegation.
Winston was not on the field with the Buccaneers on Friday ahead of Sunday's game against the Miami Dolphins. He has not practiced all week because of a shoulder injury.
The Uber driver told BuzzFeed News that she picked up Winston in Scottsdale, Ariz., at around 2 a.m. local time on March 13, 2016. The driver alleged Winston "behaved poorly" as soon as he got in the car, and then asked her to stop at a drive-thru restaurant during the ride.
While in line, the driver said Winston, who was the only passenger in the car, reached over and "grabbed" her crotch for three to five seconds.
The driver told BuzzFeed News that she reported the incident to Uber at the time. An Uber spokesman told ESPN that the "behavior the driver reported is disturbing and wrong. The rider was permanently removed from the app shortly after we learned of the incident."
Winston was in Arizona for Kurt Warner's annual "Ultimate Football Experience" event during the weekend that the incident allegedly occurred.
Winston denied that accusation in a statement posted to social media on Friday afternoon.
"A news organization has published a story about me regarding an alleged incident involving a female Uber driver from approximately two years ago," Winston said in the statement. "The story falsely accuses me of making inappropriate contact with this driver. I believe the driver was confused as to the number of passengers in the car and who was sitting next to her. The accusation is false, and given the nature of the allegation and increased awareness and consideration of these types of matters, I am addressing this false report immediately. At the time of the alleged incident, I denied the allegations to Uber, yet they still decided to suspend my account.
"I am supportive of the national movement to raise awareness and develop better responses to the concerns of parties who find themselves in these types of situations, but this accusation is false. While I am certain that I did not make any inappropriate contact, I don't want to engage in a battle with the driver and I regret if my demeanor or presence made her uncomfortable in any way."