More than 100 workers at Highway 407 ETR hit the pavement Wednesday morning.
The strike by the 105 customer service and call centre workers represented by the Canadian Auto Workers Local 414 followed weeks of negotiations.
The workers began picketing outside the company's head office in Woodbridge, just north of Toronto at 7 a.m.
The strike is not expected to affect traffic on the toll road that stretches across the Greater Toronto Area from Oakville to Pickering.
The ETR office is also expected to remain open, but hours of service in the office could be reduced.