Objectives : To clarify (1) the prevalence and associating factors of worklife conflict (WLC) ; (2) the details of genderbased discrimination ; and (3) the association between WLC and genderbased discrimination among various professionals in a medical university organization. Methods : This crosssectional study, conducted in 2017, included all employees working at a public medical university and two affiliated hospitals that lie in provincial cities in Japan. The outcome of interest was timebased WLC in the worktofamily or familytowork direction, measured with a shortened version of an existing scale. Genderbased discrimination was measured according to a threepoint scale. Results : Among the 3,347 employees, complete data sets were available for 2,285 (complete response rate, 68.3%). Of these, approximately 30% of respondents had perceived WLC. Multivariable logistic regression analysis showed that faculty members, nurses, and employees between 30 and 39 years old had a greater risk of WLC regardless of gender. Men were more likely to perceive genderbased discrimination in the contents of their work and the number of incidental tasks, while women were more likely to perceive discrimination with promotions and evaluation of academic achievements. Both men and women respondents who perceived genderbased discrimination had an increased risk of WLC. Conclusions : When promoting organizational wellbeing in a medical university, increased attention should be paid to faculty members, nurses and employees between 30 and 39 years old, as they have a greater risk of WLC. Our results also suggest that promoting gender equality is important to help achieve appropriate worklife balance.