The project has attracted criticism from environmental and tribal groups.
"We cannot continue to legitimize and provide cover for the MPCA's war on black and brown people," their resignation letter states.
Honor the Earth filed the petition with the Minnesota Public Utilities Commission Tuesday.
The Enbridge pipeline replacement project would replace a crude oil pipeline in northern Minnesota.
The move came as the legislature began its fourth special session.
The action is the latest in a year-long process surrounding the controversial project.
The Supreme Court declined to take up a challenge against the project.
It's a setback for pipeline company Enbridge.