Walker said he was simply educating voters about Burke's record at the company started by her father in the 1970s that has grown into the world's second-largest bike manufacturer. Burke worked there twice, leaving in 2004.
"We're not criticizing Trek, we're pointing out that voters deserve to know the full record," Walker said during a stop at Milwaukee Area Technical College in Oak Creek.
Burke, who has touted her ties to Trek throughout the campaign, released a response ad Friday which accused Walker of launching "an outrageous attack on a great Wisconsin company." Her brother, Trek president John Burke, on Thursday called Walker's ad "blatantly false" and said he should take it off the air.
Walker has refused and stood by the spot again Friday.
"She personally profits from a company who took state taxpayers' money, sent jobs overseas and part of those jobs went to places like China where they make less than $2 an hour," Walker said Friday, parroting language in his ad.
"You didn't hear an editorial on my part, I just simply stated the facts," Walker said.
Trek employs 1,000 people in Wisconsin and 800 people outside of the country, including at manufacturing plants in China, Germany and Holland. John Burke told The Associated Press in an interview Thursday that the company follows all local labor and wage laws.
"We produce product in a lot of places and there are different wages for different jobs," he said when asked whether workers at the China plant earned $2 an hour. "We always pay in accordance to labor guidelines and rules and what's the standard wage for the area. We're very concerned for workers' rights and the workers' environment."
Walker called those comments vague and said Trek has yet to refute claims in his ad that workers in China are paid $2 an hour.
"If Trek Bicycles' management wants to come out and clarify that somehow we're wrong, they're paying more than that, if they want to dispute those facts, that's fine," the governor said.
John Burke also said that he, not his sister, made the decisions about where to locate jobs.
"It's interesting to hear the CEO of Trek say that Mary Burke had nothing to do with manufacturing at a company that manufactures bikes because that might suggest to some that maybe she's exaggerating her role in the company," Walker said.
Mary Burke worked as the head of Trek's European division from 1990 to 1993. During that time, she said she expanded sales from $3 million to $50 million. She also worked for Trek from 1995 until 2004, with her most recent job being director of planning and strategic planning.
Burke has not disclosed her net worth, but mandatory filings with the state show she owns at least $50,000 in Trek stock. She has already spent $400,000 of her own money on the race.
Before Burke got into the race, Walker's economic development agency in 2012 used Trek in a marketing campaign designed to lure more business to the state. John Burke was featured prominently in a promotional video created by Walker's administration.
Walker said Friday voters "deserve to know everything about Trek and to make their judgment."
Bauer reported from Madison, Wisconsin.