The inclusive Robins will wear their colourful new on-off shirt in the National League North when they Bradford (Park Avenue) this weekend.
They are keeping their famous red and white stripes at home to don the trademark LGBT colours - and plan to raise money in the process.
Alty hope the kit can inspire a big performance as they look to enhance their play-off bid by winning for the third time in four games.
Manager Phil Parkinson knows the club are sending out a hugely important message - and says his players are fully behind taking to the field in the specially-made rainbow kits.
Director Bill Waterson said: “We are extremely proud to be able to play a part in Football v Homophobia month.
“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 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.