The public address announcement to the raucous, but good-natured, crowd at the first tee came after a statement issued on Sunday by the PGA of America.
"Rooted in the ideals of sportsmanship and international goodwill, the Ryder Cup is about having a spirited passion for not only the victory, but for the game of golf," it read.
"We are encouraging all spectators on Sunday to be passionate and support their team in a way that is respectful to those around them, the players, and the Ryder Cup.
"Our security staff will continue to enforce a zero tolerance policy, removing from the course any fans who are disruptive in any way, including the use of vulgar or profane language directed at the players."
The first two days of the three-day competition were marked by numerous outbursts directed at European players that were often disruptive, in bad taste and flew in the face of golf etiquette.
The offenders were in the distinct minority among the 50,000 fans drawn to Hazeltine for golf's most celebrated team match play competition, cheering heartily for the home side in their campaign to end a string of three European wins in the biennial event.
The scene at the first time was boisterous and full of fun, with duelling chants and sing-songs from the opposing supporters.
"I Believe That We Will Win," chanted American fans, once they tired of the familiar "USA, USA", as Patrick Reed took the first tee for his match against Rory McIlroy.
The European contingent in the packed grandstand countered with, "Rory's on Fire, Patrick Reed is Terrified," which set the Northern Irishman golfer into a jig that matched the rhythm.
The U.S. side carried a 9-1/2 to 6-1/2 lead into the decisive session of 12 singles matches worth a point each.
The Americans needed five more points to claim the golden trophy for the first time since 2008, while Europe required seven and a half points to retain the Cup.
(Reporting by Larry Fine, editing by Pritha Sarkar)
