Canadian drivers (CN), trained people and yard staff notified the strike before Tuesday's deadline.
The union, which represents 3,200 workers, provided a 72-hour notice on Saturday, while contract negotiations continue this weekend.
The Teamsters Canada Rail Conference warned in October that it was prepared to commence work after more than six months of unsuccessful negotiations.
A strike can begin at 12:01 ET on Tuesday, now that the warning has been given.
Workers, especially in Canada's major urban centers, have been without a contract since July 23.
Rob Reilly, executive vice president and chief operating officer of CN, said in a statement that the company will continue to trade "in good faith" in the coming days.
"If no agreement can be reached this weekend, we will once again encourage the union leadership to accept binding arbitration as an alternative to disrupting the Canadian economy."
The strike warning comes a day after the Montreal CN confirmed job cuts, saying it was "adjusting its resources to demand." The CN would not say how many people will be affected.