The two met face-to-face earlier today for the first time since Article 50 was last week triggered by the UK.
And May took the opportunity to press Tusk on the future of the British territory after the EU last week suggested that Gibraltar could be excluded from any Brexit deal without an independent agreement between Spain and the UK.
A Downing Street spokesman said May had made clear the UK's position on the Rock had not changed, stressing that "the UK would seek the best possible deal for Gibraltar as the UK exits the EU and there would be no negotiation on the sovereignty of Gibraltar without the consent of its people."
It comes after foreign secretary Boris Johnson promised "implacable" and "rock-like" support for the British territory, which voted strongly to Remain in the EU in last year's Brexit vote.
May and Tusk also discussed the upcoming Brexit negotiations, which will begin in earnest next month.
“[May] said the UK looked forward to formally beginning negotiations once the 27 Member States agreed guidelines," Downing Street said.
"Both leaders agreed that the tone of discussions had been positive on both sides, and agreed that they would seek to remain in close touch as the negotiations progressed."