Context Research suggests that athletic training students lack knowledge and experience providing care to transgender patients. Additionally, research has identified a lack of comfort with sexual health screening in peer health professions. Objective To assess how a curriculum, including a standardized patient (SP) encounter, influenced attitudes and skills in working with sexual health and gender minorities. Design Prospective observational study. Setting Simulation lab. Patients or Other Participants Twenty cisgender postbaccalaureate professional athletic training students (females = 16, males = 4; age = 23 ± 2 years). Intervention(s) The intervention included a focused curriculum on transgender health care and sexual health. In a culminating SP encounter, one group (n = 10) interacted with a cisgender woman and the second group (n = 10) with a transgender woman. Main Outcome Measure(s) The students completed a postintervention survey. Instruments included the Attitudes Towards Transgender Patients tool, which is divided into 3 subscales: clinician education, transgender sport participation, and clinician comfort; and the the Sexual Health Knowledge and Attitudes and Sexual History–Taking instruments, which evaluated the effectiveness of the sexual health curriculum on knowledge, attitudes, and comfort. The investigator and SP actor evaluated the SP encounters. Data were analyzed using descriptive statistics, nonparametric Mann-Whitney U, and 1-way analyses of variance. Results We identified a significant difference between those completing a transgender SP encounter (mean = 5.30 ± 2.11) and those completing the cisgender SP encounter (mean = 3.50 ± 0.97) on the clinician education subscale (P = .035). There were no differences between groups on the transgender sport participation (P = .70) and clinician comfort (P = .32) subscales. On the SP actor evaluation, we found no significant differences (P = .08). Conclusions The curriculum and SP encounter influenced knowledge, attitudes, and comfort when working with gender minorities and screening for sexual health.