Communists and other groups that opposed President Viktor Yushchenko in a long and bruising campaign leading to his election last year joined together to vote down the proposal.
Yushchenko, bolstered then by "Orange Revolution" rallies directed against his predecessor Leonid Kuchma, has predicated virtually every policy move on plans to join both organisations.
The vote to ratify a memorandum on the airlift plan, signed in June 2004 while Ukraine was still led by Kuchma, received 207 votes in the 450-seat chamber - short of the required 226.
"This concerns Ukraine taking part in air transport and movement of troops to any place on earth in support of Nato operations in crises and emergencies," said Hryhory Kryuchkov, Communist head of parliament's Defence Committee.
"This could lead to Ukraine being dragged into these conflicts even without being a Nato member."
Defence Minister Anatoly Hrytsenko told the chamber there were good economic reasons for the memorandum as Ukraine would be paid for the use of its fleet of heavy aircraft.
Yushchenko's administration has suggested Ukraine might help with its airlifting capacity in a number of major Nato operations, including Afghanistan.
The president announced Ukraine's intention to seek Nato membership while visiting its Brussels headquarters in February, though opinion polls show most Ukrainians do not favour membership of the alliance.
Members say the reluctance is linked to latent hostility based on Soviet-era negative images of Nato.
Nato has agreed to help upgrade Ukraine's defences and ensure the country meets the necessary standards but has refused to give any indication of when it might be offered membership.