"The security and stability of the Black Sea are in the US's national interest and are critical to the security of NATO's eastern flank," Austin said.
US defence secretary Lloyd Austin visited Ukraine on Tuesday, underlining Washington's support for the country's sovereignty amid tensions with Russia.
It's part of a trip that includes a visit to Georgia on Monday and Romania on Wednesday ahead of a NATO meeting in Brussels.
"We again call on Russia to end its occupation of Crimea, to stop perpetuating the war in Eastern Ukraine, to end its destabilising activities in the Black Sea and along Ukraine borders, and to hold its persistent cyberattacks and other malign activities against the US and its allies and partners," Austin said.
Since 2014, the ongoing war between Russia and Ukraine has seen around 14,000 Ukrainians killed by Russian forces around the Donbas region in Eastern Ukraine.
Ukraine's President Volodymyr Zelenskyy hailed the U.S. as the country's “chief partner in security and defence” during a meeting with Austin.
Russian Foreign Minister Sergey Lavrov on Monday announced it was suspending its mission to NATO.
There have been growing tensions between Moscow and the defensive alliance after several members of Moscow's delegation were expelled for alleged spying.
In Romania on Wednesday, Austin said that the stability of the region around the Black Sea was important to the US.
"The security and stability of the Black Sea are in the US's national interest and are critical to the security of NATO's eastern flank. The region, it goes without saying, that the region is vulnerable to Russian aggression, and we've seen evidence of that by ongoing actions in eastern Ukraine, the occupation of parts of Georgia and the militarisation of the Black Sea and provocative actions in the air and at sea," he said.
"I would go further to say that Russia's destabilizing activities in and around the Black Sea reflect its ambitions to regain a dominant position in the region and to prevent the realization of a Europe that's whole, free and at peace."