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Background: Transgender and gender diverse people (TGD) often turn to online platforms for information and support regarding gender-affirming hormone therapy (GAHT), yet analysis of this social media content remains scarce. Objective: We characterize GAHT-related videos on TikTok to highlight implications relevant to GAHT prescribers. Methods: We used a web-scraper to identify TikTok videos posted under the hashtags #genderaffirminghormonetherapy and #genderaffirminghormones as of November 2023. We identified recurrent themes via qualitative content analysis (QCA) and assessed health education videos with the Patient Education Materials Assessment Tool for Audiovisual Materials (PEMAT-A/V) scale and a modified Currency, Relevance, Authority, Accuracy, and Purpose (CRAAP) test. Results: Out of 69 videos extracted, 71% were created by GAHT users, 24.6% were created by healthcare workers, and 21.7% were created to provide health education. Themes included physical changes on testosterone, GAHT access, and combating misinformation and stigma surrounding GAHT. Health education videos scored highly on PEMAT-A/V items assessing understandability (mean 88.3%, standard deviation (SD)+11.3, and lower on actionability (mean 60.0%, SD+45.8). On the CRAAP test, videos scored highly on the relevance, authority, and purpose domains, but lower on the currency and accuracy domains. Conclusions: Discussions of GAHT on TikTok build community among TGD users, provide a platform for digital activism and resistance against legislation that limits GAHT access, and foster patient-provider dialogue. Educational videos are highly understandable and created by reliable sources but vary in terms of currency and quality of supporting evidence, and lack in actionability.