HMCS Fredericton — which has recently been upgraded to better track and target aircraft — will take over from HMCS Toronto in the operations in the Mediterranean Sea.
Toronto has been in the area since August and Fredericton is expected to deploy for a similar six-month period after the ships meet up in Spain.
Defence Minister Rob Nicholson said the ship is being dispatched as part of a broader Canadian message to Russian President Vladimir Putin that Ottawa disapproves of his government's involvement in the Ukraine.
"Whether it be with the deployment of this ship, with the deployment of our air force and the soldiers we have had on the ground we are absolutely committed to sending out the message that what Putin has done is unacceptable," the minister said after the departure ceremonies.
Four CF-18s were deployed last fall as part of NATO's mission in response to Russia's annexation of Crimea and support of pro-Moscow backed rebels in eastern Ukraine.
Nicholson has recently said Canada is searching for bilateral ways to expand military training with the embattled country. Canada has already donated quantities of non-lethal military equipment to the government in Kyiv.
In August, the Defence Department delivered helmets, protective eyewear, first-aid kits, tents and sleeping bags. Last month, the Canadian Forces began shipping more gear, including tactical communication systems, explosive ordinance disposal equipment, tactical medical kits, night vision goggles and winter clothing.
Fredericton is the first frigate to participate in the NATO operation that has received upgrades through a $4.3-billion modernization program of the warships.
The ship has a new combat management system, refurbished radar systems, upgraded communications and a more recent generation of missiles.
Rear Admiral John Newton said the ship has received significant improvements in terms of its ability to detect air threats and then transfer that information into its own defence systems.
"There's a very quick turnover from tracking to locking on with missile control and gun control radars," he said.
In a recent interview with The Canadian Press, NATO's supreme commander has acknowledged that Russian air activity throughout eastern Europe has steadily increased.
The ship departed with a crew of about 250 people and a CH-124 Sea King helicopter and air attachment on board.
The Royal Canadian Navy hasn't indicated yet how ships on overseas missions will be refuelled and resupplied without the use of the two Canadian supply ships — which are both out of service.
Nicholson said Canada is working with its allies, but said no firm decision has been taken yet on what to do about the supply ship issue.
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