Russia's Foreign Ministry announced the expulsions earlier in the day, in retaliation to a similar step by NATO last week.
"Canada strongly objects to Russia's unjustifiable decision to expel two Canadians serving at the NATO Information Office in Moscow," Foreign Affairs Minister Lawrence Cannon said, adding that he would seek "a full explanation from the Russian ambassador."
"I find it rather strange that this has happened considering that NATO has re-engaged with Russia," he said.
Last week, NATO cancelled the accreditation of two Russian representatives at the Western alliance's Brussels headquarters, in connection with a spying scandal in Estonia.
The Russian Foreign Ministry said earlier on Wednesday that Canadian Ambassador Ralph Lysyshyn had been summoned to the ministry, and informed that the expulsions were "a response to an unfriendly act on the part of NATO in relation to staff at the Russian mission to NATO."
The Russian decision applies to "director of the NATO Information Office in Moscow, Canadian Embassy attache Isabelle Francois, and a second bureau official, Canadian Embassy attache Mark Opgenorth," the ministry said.
NATO has called Russia's decision "very unfortunate and counterproductive".
In late February, a former head of security at the Estonian Defense Ministry was jailed for selling secret NATO documents to Russia. Investigators said Herman Simm passed nearly 3,000 documents to Russian intelligence.
Relations between Russia and NATO, which were broken off after last August's war between Russia and Georgia, have been further strained by NATO-led military exercises which began on Wednesday in Georgia.
Russia has warned that the Cooperative Longbow/Cooperative Lancer 2009 command-and-staff exercise could encourage President Mikheil Saakashvili's regime to carry out new acts of aggression.
