Background: This study examined the relationship of academic performance with the Big Five traits of personality, academic motivation, and gender in a cross-cultural context. Methods: Participants in the study were 424 university students of physical education (PE) departments from Poland (53%) and Ukraine (47%). Undergraduates completed a brief version of the International Personality Item Pool (Mini-IPIP) to assess the Five-Factor model of personality, the Academic Motivations Scale (AMS), and grade point average (GPA). Results: Polish PE students scored higher in emotional stability and extroversion and had a higher GPA than Ukrainian PE undergraduates. Gender differences were found in both personality traits and academic motivation scales. Intrinsic motivation may predict academic performance. Conscientiousness and intellect emerged as mediators of the relationship between intrinsic motivation and academic performance and gender was found as a moderator in the relationship between conscientiousness and academic success. Conclusions: Women are more motivated regarding academic achievements than men. In addition to intrinsic motivation, the most important factors for academic grades are some personality traits, gender, and cultural differences. Openness and conscientiousness in men are mediators between intrinsic motivation and academic performance. The results of this study may be useful for PE academic teachers to improve the motivation of their students.