The introduction of emtricitabine/tenofovir diphosphate (FTC/TDF) as pre-exposure prophylaxis (PrEP) for HIV prevention has raised questions regarding which clinicians will serve as prescribers and how providers will be educated about this HIV prevention strategy. We piloted an HIV Prevention Education Program called PrEP University (PrEP U) to address knowledge gaps in HIV prevention among medical trainees. We examined PrEP awareness and assessed learning as a result of the program, measuring knowledge before and after the lectures with an anonymous 5-question multiple choice test. A total of 198 learners participated in PrEP University, which included 127 first year medical students, and post-graduate trainees in internal medicine (n = 23), family medicine (n = 16), OBGYN (n = 13) and pharmacy (n = 19). Prior to PrEP U, 27% of all participants were not aware of PrEP and an additional 8% were unsure if they had heard of it. Knowledge increased significantly after the education program among trainees in OBGYN (2.3 vs 3.8, p < 0.001), pharmacy (1.4 vs 2.5, p = 0.012) and school of medicine (3.3 vs 4.4, p < 0.001), with a trend seen in family medicine (2.7 vs. 3.7, p = 0.067) and internal medicine (2.7 vs 3.4, p = 0.068). Overall, an HIV Prevention Education Program was successfully administered to nearly 200 participants and resulted in improved knowledge of HIV prevention and PrEP across. Pharmacists and OBGYN physicians are two groups with an expanding role in the use of PrEP. Similar programs at other medical schools should be implemented to ensure that future physicians and pharmacists are comfortable with PrEP prescription.