Roy Moore, 69, violated judicial ethics with an order seen as directing probate judges to deny marriage licences to gay couples, a judicial panel ruled.
The decision was a "politically motivated effort" by radical groups, he said. His lawyer has vowed to appeal.
It is the second suspension for Mr Moore, an outspoken conservative.
In 2003, he was removed for refusing to take down a monument of the Ten Commandments he installed at a state building.
He was re-elected as chief justice of the state's Supreme Court in 2012.
In Friday's decision, the nine-member Alabama Court of the Judiciary unanimously decided to suspend him for the remainder of his term without pay.
The move essentially removes Mr Moore from the bench, as he will be unable to seek re-election at the end of his term, in January 2019, because of age restrictions, his lawyer Mat Staver said.
Reacting to the decision, Mr Moore said in a statement: "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."
The panel found that Mr Moore's ruling in 6 January showed "disregard for binding federal law" after the US Supreme Court landmark decision in June 2015 affirming gay marriage rights.
Testifying in his defence, Mr Moore said there was uncertainty after conflicting opinions on gay marriage from state and federal courts.
Mr Moore is known for his opposition to same-sex marriage, and has called homosexuality an "inherent evil" in the past.
His suspension was celebrated by the civil rights group that filed the complaints in 2003 and 2016.
"Moore was elected to be a judge, not a preacher," Richard Cohen, president of the Southern Poverty Law Center, said in a statement.
"The people of Alabama who cherish the rule of law are not going to miss the Ayatollah of Alabama."
Alabama's top judge has been suspended for the remainder of his term for defying federal court rulings that legalized same-sex marriage.