BACKGROUND Transgender student-athletes are increasingly participating in sport, requiring athletic trainer (AT) preparedness to care for their needs. OBJECTIVE To measure ATs' (1) perceived definition of transgender, (2) comfort and competence working with transgender student-athletes, (3) sources of education, (4) perceived legal concerns, and (5) perception of competitive advantage. METHODS Cross-sectional study. METHODS Mixed-methods survey. METHODS Collegiate or university ATs (n = 5537) received an email invitation to participate; the data of 667 ATs were included in the analysis. METHODS A multipart 43-item questionnaire addressing the primary objectives of the study, with other factors that were explored in relation to these objectives to uncover potential influences on their responses. We calculated descriptive statistics, and for open-ended responses, we used the consensual qualitative research tradition. RESULTS About half (48.1%, n = 321) of the participants agreed they were competent in treating transgender patients, but only 36.0% (n = 240) believed they were competent in practicing collaboratively with an endocrinologist in the drug-screening processes. Fewer than half (45.6%, n = 304) of participants felt they were competent in using appropriate terminology relating to transgender patients. The ATs disagreed when asked if they were competent regarding counseling transgender patients about the effects of hormone replacement therapy on sport participation (48.1%, n = 321) or on mental health concerns (40.3%, n = 269). Participants learned most frequently from media outlets (35.2%, n = 235) or personal experiences with family, friends, or themselves (33.7%, n = 225), yet 35.1% (n = 243) received no education in caring for transgender patients. Many ATs (41.2%, n = 278) believed that transgender female student-athletes had a competitive advantage. In contrast, 6.6% (n = 44) of participants indicated that transgender male student-athletes had a competitive advantage. CONCLUSIONS Although collegiate ATs generally felt competent in treating transgender patients, they did not feel capable of addressing specific aspects of transgender patients' health care needs. Regardless of the resulting perceived unfair advantage, ATs must be aware of the regulations and therapeutic effects associated with hormone-related therapy for transgender student-athletes.