Background: Epidemiologic studies provide broad-based evidence that males are at greater risk of severe periodontal diseases than females. Our recent findings further revealed that male gender was an independent risk factor for gingival bleeding in late adolescents in Japan. Gingival health status has been reported to be affected by oral hygiene behavior. However, gender difference in this regard has not yet been clarified. Methods: We conducted a retrospective review of mandatory medical questionnaires administered as part of a legally required freshman medical checkup between April 2017 and 2019 at the University of Tokyo. Results: Among a total of 9376 sets of responses, chosen subjects were 9098 students aged 17–19. For frequency of daily brushing, males brushed less frequently than females (p < 0.001): 1 time or less (male: 22.9%, female: 11.2%), twice (65.0%, 69.2%), 3 times or more (12.1%, 19.6%). For the duration of brushing each time, males brushed for a shorter period of time than females (p = 0.005): 1 min or less (male: 17.2%, female: 14.1%), 2–3 min (46.9%, 49.2%), 4 min or more (35.9%, 36.7%). (1) Male gender, (2) lower frequency of daily brushing and (3) shorter duration of brushing each time, were significantly associated with the presence of gingival bleeding (p < 0.001 for all). Multivariate regression analysis showed that: (1) male gender (odds ratio 1.29, 95% confidence interval 1.15–1.44); (2) frequency of daily brushing: 1 time or less (2.36, 2.02–2.76), twice (1.45, 1.27–1.67); and (3) brushing duration each time: 1 min or less (1.57, 1.39–1.78), 2–3 min (1.26, 1.14–1.39), were independent risk factors for gingival bleeding (p < 0.001 for all). Conclusions: Males showed poorer oral hygiene behavior than females in late adolescents in Japan. Male gender was an independent risk factor for gingival bleeding, as well as poor oral hygiene behavior.