Chief Justice Roy Moore said his Christian faith meant he had to direct local officials not to issue marriage licenses to same-sex couples.
In a statement he said his sacking was a "motivated effort by radicals" because of his "opposition to their immoral agenda".
The Supreme Court of the United States passed a law in June 2015 that meant every state would have to legalise gay marriage. It has faced resistance in more conservative states.
Moore has been suspended without pay for the rest of his term. It means he has effectively been sacked as he will be too old to seek re-election at the term end, in January 2019.
Officials said he had violated judicial ethics with his guidance, which suggested local authorities could not issue marriage licences to gay couples.
He plans to appeal.
Moore, 69, has been suspended before. In 2003 he rejected a federal order to take down a Ten Commandments monument he had installed in a state court.
"This was a politically motivated effort by radical homosexual and transgender groups to remove me as Chief Justice of the Supreme Court because of outspoken opposition to their immoral agenda," he said.
His lawyer, Mat Staver, called it a "de facto removal".
Civil rights groups welcomed the move. The Southern Poverty Law Centre's Richard Cohen said: "The people of Alabama who cherish the rule of law are not going to miss the Ayatollah of Alabama.
"It undermined the integrity of the judiciary, the spectacle of a chief justice telling other judges not to follow a court order."
