Russia reacted quickly to the move, calling it an "unfriendly step" that will only "add to nuisances" in ties.
The BBC's David Stern in Kiev says it is not clear when Ukraine will apply for Nato membership and many officials see it as a distant prospect.
Ukrainian President Petro Poroshenko pledged to seek Nato membership over Russian support for rebels in the east.
Russia denies supplying the rebels with weapons but is subject to EU and US sanctions over the crisis.
Ukraine's MPs overwhelmingly backed the move, by 303 votes to eight.
The non-aligned status, which Ukraine adopted in 2010 under Russian pressure, prevents states from joining military alliances.
Addressing foreign ambassadors on Monday night, President Poroshenko said Ukraine's "fight for its independence, territorial integrity and sovereignty has turned into a decisive factor in our relations with the world".
In a Facebook post on Monday, Russian Prime Minister Dmitry Medvedev warned that Ukraine's rejection of neutrality would have "negative consequences".
"In essence, an application for Nato membership will turn Ukraine into a potential military opponent for Russia," he wrote.
A Nato spokesman in Brussels said on Tuesday that any accession to the alliance would probably take years, Reuters reported.
