BACKGROUND: Maintenance of good health and a healthy lifestyle have significant impacts on the lives of university students. However, university students are prone to engage in risky health behaviors, resulting in impaired health status. Electronic health (eHealth) literacy is an important factor in maintaining a healthy lifestyle. However, no studies have assessed the eHealth literacy levels and the associated lifestyle behaviors among university students in Japan. OBJECTIVE: The purposes of this study were to clarify the eHealth literacy level, the participant characteristics associated with eHealth literacy, and the association of eHealth literacy with lifestyle behaviors of students in a Japanese university. METHODS: A questionnaire-based cross-sectional study of 3183 students at a national university in Japan was conducted. eHealth literacy was quantified using the Japanese version of the eHealth Literacy Scale (eHEALS). The association between participant characteristics (gender, school year, department of study, and living status) and eHEALS score was assessed using t tests. Additionally, the associations of eHealth literacy with lifestyle behaviors (exercise, smoking, alcohol consumption, etc.) were evaluated using logistic regression analyses. RESULTS: The mean eHEALS score was 23.6/40 points. The mean eHEALS score for students in medical departments was 27.0/40 points, which was 2.9 points higher than that of nonmedical students (P<.001). Similarly, the graduate school participants had higher scores than the undergraduate students. The proportion of participants who exercised regularly was higher in the high eHEALS score group than in the low score group, with an adjusted odds ratio of 1.39 (P<.001). CONCLUSIONS: The eHealth literacy level of university students in Japan was comparable to that of the general Japanese population. Graduate students, as well as those in medical departments, had higher eHealth literacy. Furthermore, students with higher eHealth literacy had better exercise routines.