Cardiff boss Matt Sherratt will be in charge on an interim basis for the rest of the Six Nations campaign, which Wales began with defeats against France and Italy.
Howley, a coaching mainstay during Gatland’s first reign in charge of Wales, returned to the staff as an assistant ahead of last year’s Six Nations.
Rob Howley will not be part of Wales’ coaching team for Guinness Six Nations fixtures against Ireland, Scotland and England.
Gloucester backs Gareth Anscombe and Max Llewellyn, who were surprise omissions from Gatland’s Six Nations squad, are now back in the fray.
Sherratt, who takes over the squad following Cardiff’s United Rugby Championship clash against Connacht on Saturday, has called up three players.