Objective: HIV-related stigma is one of the strongest barriers to prevention and treatment. HIV prevalence in U.S. college students is estimated around 0.02%, but is thought to be drastically underreported. We examined the influence of HIV knowledge on the relationship between HIV testing history and stigma in college students. Participants: A random sample of 2343 students, over the age of 18, attending a large university in the southeastern United States completed the survey in January 2016. Methods: A mediation model was constructed in regression framework to explore the relationship between HIV testing history, knowledge, and stigma. Results: HIV testing history was associated with higher knowledge scores (a path: B = 4.08, p < 0.001) and higher knowledge scores were associated with lower stigma (b path: B = 0.01, p < 0.001). These results suggest that HIV knowledge partially mediates the relation between HIV testing history and stigma in college students. Conclusions: HIV testing history may decrease stigma by increasing knowledge. Results can be used to inform college health promotion practice on developing programs and services.