Objective: College students make many important decisions related to sexual health, but little is known about their reproductive health knowledge. The purpose of the study was to examine health literacy and knowledge of female reproduction, contraception, and sexually transmitted infections (STIs) in college students. Participants: Undergraduate students (n = 323) completed a survey in November 2017. Methods: The survey included the Newest Vital Sign (NVS), Knowledge of the Female Body (KFB) scale, contraceptive questions, STI prevention questions, and sexual history questions. Results: NVS and KFB scores were positively correlated (p < 0.01). Male participants demonstrated significantly lower KFB scores than female participants (p < 0.001). Transgender participants demonstrated lower health literacy and lower knowledge of basic female reproduction than cisgender participants. Participants demonstrated low knowledge of the menstrual cycle, early physical signs of pregnancy, and certain contraceptives. Conclusions: Undergraduate students have major reproductive knowledge gaps. Healthcare providers need to consider health literacy and knowledge level when educating college students.