Boris Johnson has warned Vladimir Putin that further incursion into Ukraine would a "tragic miscalculation" and expressed "deep concern" at Russian activities.
The Prime Minister spoke to Mr Putin on Wednesday afternoon after being forced to delay the call to answer questions about the Downing Street partygate saga.
Downing Street said Mr Johnson stressed during a call lasting around 45 minutes that Ukraine has a right to aspire to Nato membership, a move the Russian President opposes.
But No 10 said the pair "agreed that aggravation was in no-one's interest" amid mounting fears of conflict in the region.
Earlier in the day, a Kremlin spokesman mocked the PM and ridiculed the "stupidity and ignorance" of British politicians.
A Downing Street spokeswoman said: "The Prime Minister expressed his deep concern about Russia's current hostile activity on the Ukrainian border.
"He emphasised the need to find a way forward which respects both Ukraine's territorial integrity and right to self-defence.
"The Prime Minister stressed that any further Russian incursion into Ukrainian territory would be a tragic miscalculation.
"The Prime Minister underscored that, under Nato's open door policy, all European democracies have a right to aspire to Nato membership.
"This right fully applies to Ukraine. He also reiterated that Nato is a defensive alliance.
"The leaders agreed that aggravation was in no-one's interest. The Prime Minister stressed the importance of dialogue and diplomacy, and the need to include Ukraine in talks."
Mr Putin has said he does not intend to order an invasion - but there are more than 100,000 Russian troops on Ukraine's eastern border, backed by tanks, artillery, helicopters and warplanes.
A senior US official confirmed the country is sending about 2,000 troops to Poland and Germany this week and roughly 1,000 more who are already based in Germany will go to Romania.
The UK is working on a beefed up sanctions regime to target Russian oligarchs and businesses - but it is not intending to send troops.
