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Enhancing communication skills is important for university students in Japan. However, a practical evidence-based approach cannot be employed for analyzing assertiveness among university students due to lack of evidence. This study examines the relationship between the experience of sports and physical activity (PA) at different developmental stages and the level of assertiveness among university students.Methods: 756 university students (235 males and 521 females) in Japan participated in this study. Experience of sports and PA levels were evaluated based on the items of sports carrier profile and the International Physical Activity Questionnaire short form. Assertiveness was assessed using the Rathus Assertiveness Schedule.Results: In the logistic regression, participation in sports at middle or high competitive levels in the elementary school stage was associated with assertiveness in university students (inexperience versus middle competitive levels: adjusted odds ratio 1.70, 95% confidence interval 1.02-2.81, inexperience versus high competitive levels: AOR 2.29, 95% confidence interval 1.11 -4.71). Moderate or vigorous PA was associated with assertiveness in university students (inactive versus moderate PA with over 720 min/wk: AOR 1.97, 95% confidence interval 1.05-3.72, inactive versus vigorous PA within 360.0-739.9 min/wk: AOR 2.19, 95% confidence interval 1.31-3.68).Conclusions: Promotion of participation in youth sports and ensuring sufficient PA levels in campus life can enhance assertiveness in university students.