BACKGROUND: Public health organizations have begun to use social media to increase awareness of health harm and positively improve health behavior. Little is known about effective strategies to disseminate health education messages digitally and ultimately achieve optimal audience engagement. OBJECTIVE: This study aims to assess the difference in audience engagement with identical antismoking health messages on three social media sites (Twitter, Facebook, and Instagram) and with a referring link to a tobacco prevention website cited in these messages. We hypothesized that health messages might not receive the same user engagement on these media, although these messages were identical and distributed at the same time. METHODS: We measured the effect of health promotion messages on the risk of smoking among users of three social media sites (Twitter, Facebook, and Instagram) and disseminated 1275 health messages between April 19 and July 12, 2017 (85 days). The identical messages were distributed at the same time and as organic (unpaid) and advertised (paid) messages, each including a link to an educational website with more information about the topic. Outcome measures included message engagement (ie, the click-through rate [CTR] of the social media messages) and educational website engagement (ie, the CTR on the educational website [wCTR]). To analyze the data and model relationships, we used mixed effects negative binomial regression, z-statistic, and the Hosmer-Lemeshow goodness-of-fit test. RESULTS: Comparisons between social media sites showed that CTRs for identical antitobacco health messages differed significantly across social media (P<.001 for all). Instagram showed the statistically significant highest overall mean message engagement (CTR=0.0037; 95% CI 0.0032-0.0042), followed by Facebook (CTR=0.0026; 95% CI 0.0022-0.0030) and Twitter (CTR=0.0015; 95% CI 0.0013-0.0017). Facebook showed the highest as well as the lowest CTR for any individual message. However, the message CTR is not indicative of user engagement with the educational website content. Pairwise comparisons of the social media sites differed with respect to the wCTR (P<.001 for all). Messages on Twitter showed the lowest CTR, but they resulted in the highest level of website engagement (wCTR=0.6308; 95% CI 0.5640-0.6975), followed by Facebook (wCTR=0.2213; 95% CI 0.1932-0.2495) and Instagram (wCTR=0.0334; 95% CI 0.0230-0.0438). We found a statistically significant higher CTR for organic (unpaid) messages (CTR=0.0074; 95% CI 0.0047-0.0100) compared with paid advertisements (CTR=0.0022; 95% CI 0.0017-0.0027; P<.001 and P<.001, respectively). CONCLUSIONS: Our study provides evidence-based insights to guide the design of health promotion efforts on social media. Future studies should examine the platform-specific impact of psycholinguistic message variations on user engagement, include newer sites such as Snapchat and TikTok, and study the correlation between web-based behavior and real-world health behavior change. The need is urgent in light of increased health-related marketing and misinformation on social media.