Nato Secretary-General Jens Stoltenberg said the two sides "disagree on the facts, the narrative and the responsibilities" on Ukraine.
Relations between Russia and the West have been tense since Russia annexed Ukraine's Crimea region in 2014.
Russia's envoy also criticised Nato for a military exercise in the Baltic.
Wednesday's talks in Brussels ran more than 90 minutes over schedule.
"Many allies disagree when Russia tries to portray [the Ukraine conflict] as a civil war," Mr Stoltenberg told a new conference.
"This is Russia destabilising eastern Ukraine, providing support for the separatists, munitions, funding, equipment and also command and control."
He added: "Nato and Russia have profound and persistent differences. Today's meeting did not change that."
Mr Stoltenberg said it was important to keep channels of communication open "to discuss our differences and to reduce the risk of military incidents".
He also said that the two sides had agreed on the importance of implementing the Minsk peace agreement, which has produced a fragile truce in eastern Ukraine.
Russia's envoy to Nato, Alexander Grushko, said a US guided-missile destroyer had sailed near the Russian port of Kaliningrad last week in an attempt "to exercise military pressure on Russia".
"We will take all necessary measures, precautions to compensate these attempts to use military force," he said.
In last week's incident, two Russian warplanes flew close to the US destroyer almost a dozen times in international waters in the Baltic Sea.
US officials described it as "an aggressive act".
The Nato-Russia Council was established in 2002. Although meetings at ambassadorial level have not taken place since June 2014, there has been other political dialogue.
Ukraine and the West accuse Russia of covertly backing the rebels who now control much of eastern Ukraine. Russia strongly denies the claims.
Nato has since moved to bolster its forces in its east European member states to counter what it says is a Russian military build-up.
Russia has described Nato's increasing presence in eastern Europe as a threat to its national security.
Differences over the conflict in eastern Ukraine have marked the first formal meeting of the Nato-Russia Council in almost two years.