Police in Windsor, Ontario, say they have commenced enforcement action at the Ambassador Bridge.
Police have started to clear a blockade of the main crossing between Canada and the United States.
After days of protests by truckers against Covid rules at the Ambassador Bridge in Ontario, officers urged them to heed an injunction against the demonstration.
The vital trade route links Windsor, Ontario, with Detroit, Michigan.
Truckers’ protests against Covid vaccine mandates are also ongoing at other border crossings and in Ottawa.
The self-styled “Freedom Convoy” movement was started by Canadian truckers opposed to a vaccinate-or-quarantine order for drivers crossing the border.
Friday’s court order against the blockade was filed by the city of Windsor and the Automotive Parts Manufacturers’ Association, which argued that it was losing as much as $50m ($39m; £29m) per day because of the convoy.
Following the injunction, Windsor Police put out a statement to “make demonstrators clearly aware that it is a criminal offence” to block the border crossing. The police added that a criminal conviction could lead to the seizure of vehicles and the inability to enter the US.
But hours later, crowds of people waving Canadian flags flouted the order and continued to occupy the bridge.
Police added on Saturday: “We urge all demonstrators to act lawfully [and] peacefully. Commuters are still being asked to avoid the areas affected by the demonstrations at this time.”
Hundreds of other protesters continue to demonstrate in the centre of Ottawa, the nation’s capital. Two other border crossings with the US are also being blocked by anti vaccine mandate protesters.
Prime Minister Justin Trudeau spoke with US President Joe Biden about the border blockades on Friday.
The week-long disruption to the bridge, which accounts for roughly 25% of US-Canada annual trade estimated to be worth $1.7bn (£1.2bn) a day, rocked the car manufacturing industry.
General Motors, Ford, Toyota and Honda plants have been forced to halt production and cancel work shifts due to parts shortages caused by the blockade.
Speaking on Friday, Mr Trudeau urged the truckers “to go home now”, saying their frustration with Covid measures had been “heard”.
“If you join the protests because you are tired of Covid, you need to understand that you are breaking laws. The consequences are becoming more and more severe,” he warned.
“You don’t want to end up losing your licence, end up with a criminal record, which will impact your job, your livelihood, even your ability to travel internationally, including to the US.”