The video release follows the release of similar efforts from other sports teams in the Bay Area, including a video from San Francisco Giants in May 2011.
The video is part of the It Gets Better Project, which aims to prevent suicide and depression in gay youth.
The 49ers are the first professional football team to create a clip for the anti-gay bullying campaign, following an online petition by local fan Sean Chapin, who collected 16,000 signatures urging the team to show LGBT support.
The one-minute video features reassuring remarks about the future from defensive tackles Ricky Jean Francois and Isaac Sopoaga, safety Donte Whitner and linebacker Ahmad Brooks.
"Something you should never experience is being bullied, intimidated, or being pressured into being someone or something you are not," Brooks says while wearing a red 49ers jersey.