The Pope was dragged into a dispute about condom distribution last year, when a top official of the Knights of Malta was fired over links between the order and charities that gave out birth control.
Francis refused to back the termination of Knights of Malta Grand Chancellor Albrecht von Boeselager, and after the battle simmered for months, the Pope asked von Boeselager's boss, Knights of Malta Grand Master Matthew Festing, to resign.
Knights of Malta spokeswoman Marianna Balfour told CNN on Wednesday that Festing, 67, agreed to step down from what would normally be a life-long position.
The Vatican said the two men met Tuesday and the Pope accepted the resignation on Wednesday.
The unusual move by Pope Francis -- forcing out the elected head of a Catholic sovereign order -- is emblematic of the clash between conservative bastions of the Church and the progressive Pope.
Condoms in Myanmar
On December 8, Pope Francis ordered an investigation into the Knights of Malta when it was revealed that Festing had fired von Boeselager.
Festing claimed that von Boeselager allowed condoms to be distributed in Myanmar through the Order's charity branch.
Von Boeselager claimed he shut down the condom distribution as soon as he had learned about it and appealed his firing to the Pope.
Festing publicly refused to cooperate with the Pope's investigation, saying the Knights of Malta are not under the jurisdiction of the Vatican.
On January 10, the Knights of Malta issued an order calling the investigation "legally impossible" and "superfluous."
The Order is legally similar to a sovereign country, has its own passports and maintains diplomatic relations with 106 states, including the Holy See and the United Nations.
Cardinal Raymond Burke, a leading conservative critic of Pope Francis, is the Cardinal Patron of the Knights of Malta. He supported Festing in the firing of von Boeselager.
In a statement Wednesday, the Vatican said a pontifical delegate will be nominated to govern the Order.
Although the Pope may not officially have jurisdiction over the Order, Festing chose to accept the Pope's request to resign, and the papal delegate signals at least a temporary takeover of the group.