The club are second from bottom of the table with just nine points from 16 games after Saturday's painful 2-1 home loss to fellow strugglers Ipswich.
"Wolves have parted company with head coach Gary O'Neil and his backroom staff," the club said in a statement.
"O'Neil arrived at Molineux just three days before the start of the 2023/24 Premier League season, taking on a significant challenge and ultimately guiding the Old Gold to a successful campaign."
Chairman Jeff Shi said the club were "grateful to Gary for all of his effort, dedication and hard work".
Just days ago Shi said the Midlands club were "united" behind the former Bournemouth boss despite Wolves' struggles.
O'Neil, 41, was appointed in August 2023 following the departure of Julen Lopetegui and signed a new four-year deal with the club in August this year.
Wolves finished 14th in his first season at Molineux but are now in a relegation scrap -- they have 11 defeats and just two wins and are four points from safety.
Frustrations boiled over in Monday's defeat to West Ham when Mario Lemina scuffled with Jarrod Bowen after the full-time whistle, resulting in the Wolves midfielder being stripped of the captaincy.
Emotions also spiked at full-time at Molineux on Saturday after Jack Taylor scored a stoppage-time winner for Ipswich.
Rayan Ait-Nouri was shown a second yellow card and Matheus Cunha had to be restrained.
After the defeat, O'Neil said he had struggled to reverse his team's slide.
"The ability of the team to cope at this level is proving unbelievably difficult for us," he told the BBC.
"I've worked at this level, this is my third year now, and I've never had so much of a struggle to help a group cope with the level -- real basic stuff."
