Ned O'Keeffe, a junior minister at the Department of Agriculture, quit after the Public Offices Commission opened an investigation involving him for an alleged breach of the Ethics in Public Office Act.
The move comes after Mr O'Keeffe voted in the Dail against a motion calling for a ban on the feeding of meat and bonemeal to all animals.
The Labour Party afterwards maintained that the minister should have declared in advance that his farm, near Mitchelstown, Co Cork, had a meat-and-bone meal licence, and therefore he had a material interest in the vote.
The Ethics in Public Office Act insists that material interests in such cases have to be declared ahead of any parliamentary vote.
Tonight Mr O'Keeffe maintained his innocence and said he was confident he would be cleared by the Commission inquiry.
But a Government source said the intervention of the Public Offices Commission had underlined the seriousness of the situation.