The aim of this prospective cohort study was to examine whether oral hygiene knowledge, and the source of that knowledge, affect oral hygiene behavior in university students in Japan. An oral exam and questionnaire survey developed to evaluate oral hygiene knowledge, the source of that knowledge, and oral hygiene behavior, such as the frequency of tooth brushing and regular dental checkups and the use of dental floss, was conducted on university student volunteers. In total, 310 students with poor tooth brushing behavior (frequency of tooth brushing per day [≤ once]), 1,963 who did not use dental floss, and 1,882 who did not receive regular dental checkup during the past year were selected. Among these students, 50, 364, and 343 in each respective category were analyzed in over the 3-year study period (follow-up rates: 16.1%, 18.5%, and 18.2%, respectively). The odds ratios (ORs) and 95% confidence intervals (CIs) for oral hygiene behavior were calculated based on oral hygiene knowledge and the source of that knowledge using logistic regression models. The results showed that dental clinics were the most common (> 50%) source of oral hygiene knowledge, and that a more frequent use of dental floss was significantly associated with dental clinics being a source of oral hygiene knowledge (OR, 4.11; 95%CI, 1.871-9.029; p < 0.001). In addition, a significant association was seen between dental clinics being a source of oral hygiene knowledge and more frequent regular dental checkups (OR, 13.626; 95%CI, 5.971-31.095; p < 0.001). These findings suggest the existence of a relationship between dental clinics being the most common source of oral hygiene knowledge and improved oral hygiene behavior in Japanese university students.