Background Youth and young adults continue to experience high rates of HIV and are also frequent users of social media. Social media platforms such as Twitter can bolster efforts to promote HIV prevention for these individuals, and while HIV-related messages exist on Twitter, little is known about the impact or reach of these messages for this population. Objective This study aims to address this gap in the literature by identifying user and message characteristics that are associated with tweet endorsement (favorited) and engagement (retweeted) among youth and young men (aged 13-24 years). Methods In a secondary analysis of data from a study of HIV-related messages posted by young men on Twitter, we used model selection techniques to examine user and tweet-level factors associated with tweet endorsement and engagement. Results Tweets from personal user accounts garnered greater endorsement and engagement than tweets from institutional users (aOR 3.27, 95% CI 2.75-3.89; P<.001). High follower count was associated with increased endorsement and engagement (aOR 1.05, 95% CI 1.04-1.06; P<.001); tweets that discussed STIs garnered lower endorsement and engagement (aOR 0.59, 95% CI 0.47-1.74; P<.001). Conclusions Findings suggest practitioners should partner with youth to design and disseminate HIV prevention messages on social media, incorporate content that resonates with youth audiences, and work to challenge stigma and foster social norms conducive to open conversation about sex, sexuality, and health.