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Background: Knowledge about the beneficial role of physical activity (PA) for health and school performance is growing. Studies investigating the link between PA and school absenteeism due to illness are lacking. Therefore, we investigated associations between habitual PA and school absenteeism due to illness in adolescents and explored whether mental health and cardiovascular fitness mediated this association. Methods: We studied 328 students in grades 7 and 9 (mean age 13.8 years; 49% boys). The PA was measured objectively by an ActivPAL3™ accelerometer attached on the thigh during 1 full week (24 hours/day). Depressive symptoms and self-esteem were self-reported by the Center for Epidemiologic Studies Depression Scale (CES-D) and Rosenberg Self-Esteem Scale, respectively, and included as a proxy for mental health in the analyses. Cardiovascular fitness was measured by the 20-m shuttle-run test. School absenteeism due to illness data was provided by the school administration. Results: The PA was not significantly associated with school absenteeism, though there was an indirect association between PA and school absenteeism by cardiovascular fitness. Conclusions: Cardiovascular fitness mediates the association between PA and school absenteeism due to illness. Thus, cardiovascular fitness of students should be improved to reduce school absenteeism due to illness.