PURPOSE: Previous studies have shown that personality traits have varying degrees of association with exercise behavior. However, the underlying mechanisms mediating this relationship remain insufficiently understood. This study aims to investigate the associations between the Big Five personality traits and exercise behavior and to construct a chain mediation model incorporating exercise self-efficacy and exercise motivation, based on the Cognitive-Affective Personality System theory, Social cognitive theory, and Self-Determination theory with the practical goal of providing guidance for promoting physical well-being among college students. METHODS: A total of 483 non-physical education majors undergraduate students (mean age = 19.71 ± 1.31 years, 56.1% female) from five randomly selected universities in Wuhan participated in the study. Data were collected through electronic questionnaires administered at two time points: baseline (Time 1; T1) assessing demographics, personality traits, and exercise self-efficacy, and four weeks later (Time 2; T2) assessing exercise motivation and behavior. The data were analyzed using SPSS 26.0 and the PROCESS macro, employing correlation and chain mediation analyses to explore the relationships among variables. RESULTS: The findings revealed that Openness, Conscientiousness, and Extraversion were positively associated with exercise behavior, Neuroticism was negatively associated, and Agreeableness showed no significant association. Mediation analysis indicated that Conscientiousness, Extraversion, and Neuroticism were related to exercise behavior through exercise self-efficacy, whereas Openness, Conscientiousness, Extraversion, and Neuroticism were related to exercise behavior through exercise motivation. Additionally, a significant chain mediation effect was observed for Conscientiousness, Extraversion, and Neuroticism, wherein exercise self-efficacy and exercise motivation sequentially mediated the relationship between these personality traits and exercise behavior. Notably, Openness did not significantly relate to exercise self-efficacy, suggesting that its relationship with exercise behavior was solely mediated by exercise motivation. Furthermore, significant gender differences were found in exercise motivation, with males exhibiting higher exercise motivation than females. CONCLUSION: This study elucidates how personality traits are associated with college students' exercise behavior through the mediating roles of exercise self-efficacy and exercise motivation. Specifically, Extraversion and Neuroticism demonstrated partial mediation through both self-efficacy and motivation, while Openness was associated with exercise behavior solely via motivation. The association between Conscientiousness and exercise behavior was fully mediated by both factors, indicating a complete indirect pathway. Although directly changing personality traits is challenging, targeting modifiable psychological mechanisms-such as enhancing self-efficacy and intrinsic motivation-may effectively promote exercise engagement. Personalized intervention strategies that account for individual personality characteristics and gender-specific motivational factors are recommended to support students' physical and mental health and reduce long-term health risks.