A video ad repeatedly criticizes the Republican for "attacking" and "assaulting" public lands since he's been in office, a veiled reference to Gianforte's attack on a reporter last year.
"Somebody's attacking our way of life and assaulting our public lands and national monuments. It's Washington politician Greg Gianforte," the ad says.
The conservation group says that they are spending $244,000 on ads through April.
Eight candidates, including six Democrats, are vying for the chance to unseat Gianforte this fall. On the eve of a special election last May, Gianforte knocked a reporter to the ground, and later pleaded guilty to a misdemeanor assault charge.
The League of Conservation Voters officials insist the ad campaign is not about Gianforte's re-election, but about bills pending in Congress.
The ad will run on video and radio streaming services, along with news and social media websites. And, users of the mapping app Waze will receive an anti-Gianforte pop-up ad when they are traveling near certain public lands in Montana, and near Gianforte's district offices.