The California Teachers Association (CTA) donated $1 million (£578,640) to the No on 8 campaign.
In November, Californian citizens will vote on Proposition 8 of the state constitution. The proposition will define marriage as existing between one man and one woman only, and will effectively ban gay marriage in the state.
The CTA’s contribution on Tuesday is the largest donation from an institution the No on 8 campaign has received.
The union also gave $25,000 to Equality for All in August, a coalition of civil rights groups, including gay rights organisations, who oppose Proposition 8.
CTA’s spokeswoman Sandra Jackson said that the union’s internal vote to oppose Proposition 8 was a large majority, and that “teachers teach the importance of equal rights for all.”