Leo and Rubio "renewed the shared commitment to fostering good bilateral relations," the Vatican said in a statement after the first meeting between the Pope and a Trump administration cabinet official in nearly a year.
Leo, the first US pontiff, drew Trump's ire after becoming a firm critic of the US-Israeli war on Iran and the US administration's hardline anti-immigration policies.
Trump responded by accusing the Pope of supporting Iran's bid to acquire nuclear weapons and also called him "weak on crime".
Ahead of his trip Rubio, a Catholic born to Cuban immigrants, emphasised common ground with the Holy See, including on the issue of religious freedom across the world.
In Cuba, he noted, the US has provided humanitarian aid with the support of the Catholic Church.
Rubio's meeting with Leo is a sign of a "strong" relationship between the Vatican and the US, State Department spokesman Tommy Pigott said.
Rubio spent two and a half hours at the Vatican before driving away in a convoy under tight security.
He also met with senior Vatican officials, including top diplomat Italian Cardinal Pietro Parolin.
The US embassy to the Holy See said on X that Leo and Rubio had discussed "topics of mutual interest in the Western Hemisphere".
The Vatican statement said the two had "exchanged views" on the world situation and spoke about "the need to work tirelessly in favour of peace".
The meeting appeared to have run longer than planned.
The Pope arrived 40 minutes late for a subsequent meeting with Vatican staffers and thanked them for being patient.
Vatican video from the beginning of the closed-door encounter showed Leo shaking hands with his guest and addressing him formally as "Mr Secretary," to which Rubio, a Catholic, responded: "Great to see you."
Rubio was also seen giving the Pope a small crystal football.
He joked that he knew that Leo, originally from Chicago and known as a fan of the White Sox, was more of a "baseball guy".
Leo gave Rubio a small pen made from wood from an olive tree, which he called "the plant of peace".
Rubio had told a White House briefing on Tuesday he expected to discuss Cuba and concerns over religious freedom around the world with Leo.
The US ambassador to the Holy See, Brian Burch, said, also on Tuesday, that the conversation between the Pope and Rubio was likely to be "frank".
Leo, who on Friday marks his first year leading the 1.4-billion-member Church, has grown more outspoken on the world stage in recent weeks.
During a four-country African tour last month he forcefully decried the direction of global leadership and said the world was "being ravaged by a handful of tyrants" in comments he later said were not aimed at Trump.
Rubio and US Vice President JD Vance, who is also a Catholic, met Leo a year ago after attending the Pope's inaugural mass.
Trump has not met the pontiff.
During his two-day visit to Rome, Rubio is due to meet Italian Prime Minister Giorgia Meloni, who has defended the Pope from Trump, on Friday.
Meloni's defence minister has said the war in Iran puts US leadership at risk.
with DPA
