Abstract Background Transgender and gender-diverse (TGD) individuals frequently turn to social media to find community, express their identities, and access essential information. These platforms are easily accessible to TGD people and enable health information–seeking in anonymous, identity-affirming spaces outside of traditional health care systems. As a result, social media has become a critical source of health information on topics like gender-affirming care for TGD individuals, specifically for TGD youth. YouTube, one of the most widely used social media platforms, is especially popular for its long-form videos made by content creators who have built dedicated followings on the platform. Among them are TGD content creators, many of whom make content documenting their medical transition and gender identity journey and provide general information about TGD topics. TGD creator content therefore makes YouTube an important platform for health education for TGD individuals. Objective This study aims to describe the health-related content shared by TGD content creators on YouTube. Specifically, we characterize the medical topics addressed, the frameworks used to discuss these topics, and the valence of creators’ health care experiences. Methods A descriptive content analysis was performed on 2485 videos posted by 42 self-identified TGD YouTube content creators. Videos were systematically evaluated for mentions of gender-affirming care and other health-related topics. We also examined whether creators framed medical information using personal narratives or an informational approach and if they characterized their medical experiences as positive, negative, or neutral. Results Most videos (n=1724, 69.4%) created by TGD content creators did not include discussions related to gender identity or transitioning. However, among the videos that did address gender identity (n=761, 30.6%), mentions of medical topics were prevalent (n=554, 72.8%). Of videos that discussed medical topics, gender-affirming surgeries (n=356, 64.3%) and hormone replacement therapy (n=307, 55.4%) were the most frequently discussed. Other commonly discussed medical topics included mental health (n=131, 23.6%) and sexual health (n=96, 17.3%). Videos covering medical topics primarily centered on personal experiences (n=411, 74.2%), with content creators often characterizing these experiences positively (n=224, 73.2%). Conclusions This study highlights the breadth of health-related information shared by TGD content creators on YouTube. Our findings underscore the role of long-form video content on YouTube as an educational resource for TGD people, offering health information that is both easy to access and grounded in lived experience. Clinicians can use these findings to better understand the health information that their TGD clients are likely to encounter online, fostering more informed and supportive conversations about gender-affirming care.