The inclusive Robins will wear their colourful new shirt in the National League North match with Bradford (Park Avenue) on February 16.
They will say goodbye to their red and white stripes and don the trademark colours - and will raise money in the process.
The match-worn tops will be auctioned off and profits will go to Pride Trust, a LGBT+ youth charity, who are rebuilding the LGBT centre in Manchester.
Passionate director Bill Waterson said: “We are extremely proud to be able to confirm that all the arrangements are in place for us to celebrate Football v Homophobia month in February by wearing this special kit against Bradford.
“We believe it will be the first time anywhere in the world that a senior football club has worn a kit solely modelled on the LGBT Pride Flag, and Altrincham will, therefore, be creating a small moment of football history.
“To us, it is a big statement. Altrincham Football Club are committed to creating a welcoming environment for all who attend our games, and our support for Football v Homophobia is part of this commitment.
“We would like to thank our main shirt sponsors, J.Davidson & Sons, for allowing their place on the front of the shirt to be replaced by the Football v Homophobia logo, and the FA, the Vanarama National League and our opponents Bradford (Park Avenue) for their support of this initiative.
“The shirts worn by our players during the game will be sold by auction after the match, and replica kits will be available for sale at the game and via our website, www.altrinchamfc.com.”