Art Pope, a philanthropist and CEO of Variety Wholesalers, said he would support Rubio's campaign in the GOP primary and in the general election.
Pope said he'd been meeting with many of the Republican candidates and made up his mind this week.
"I think Sen. Marco Rubio is both the most qualified, has the best all-around experience, and I agree with him more so on the issues than all the other candidates," Pope told CNN.
The businessman and former politician has invested millions in North Carolina politics and is an ally of the Koch Brothers' network of advocacy groups and foundations.
He wouldn't discuss any dollar amounts behind his planned support of Rubio, but pledged to "do whatever I can" to get Rubio elected, including through his contributions, advocacy for Rubio and frequent role as a national Republican Party convention delegate.
Pope said Rubio's experience as speaker of the Florida House especially appealed to him.
"To be elected by your peers, your colleagues, is a good indication of your leadership," Pope said, adding state speakers have a heavy set of policy responsibilities. "That is more experience than President (Barack) Obama had when he was a member of the Legislature in Illinois."
He also cited the senator's positions on national security, taxes, immigration and spending as being appealing.
North Carolina's primary is in mid-March, making it a more important prize this election cycle than previous ones, where it voted later in the year. That was part of Pope's reasoning for getting behind Rubio in the primary, which he sat out last election cycle.
It's the latest victory for Rubio in the GOP donor race, where he's competing against other big fundraisers, including Texas Sen. Ted Cruz and former Florida Gov. Jeb Bush, who has been sinking in the polls.
Rubio has been endorsed by other mega-donors including Ken Griffin, Paul Singer and Frank VanderSloot.