The tainted vote led to Russia's annexation of the strategic Crimean peninsula and heated up a crisis which persists today.
In a statement, Nicholson said Canada's position on the issue remains firm.
"Canada will never recognize the illegal annexation of Crimea," he said.
He urged Russian President Vladimir Putin to withdraw from Ukraine.
The Ukrainian issue is an important one for Prime Minister Stephen Harper. He has visited Kyiv to show support and last September invited Ukrainian President Petro Poroshenko to address a joint session of Parliament.
Harper has also spoken strongly about it on the international stage.
Last November, at the G-20 meeting in Australia, Harper was uncompromising, saying a major power can't just seize sovereign territory.
"We send the message, as I think we have in Canada, that whether it takes five months or 50 years, we're not going to drop the subject until Ukrainian territory is returned to Ukrainians."
In addition to imposing a series of sanctions against individuals and entities in both Russia and Ukraine, Canada has provided non-lethal military equipment to the Ukrainian government and provided humanitarian support and assistance to civilian population.
More than 6,000 people have died during the unrest.
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