A surprisingly high prevalence of vitamin D (vit D) deficiency has recently been reported worldwide. This study investigated the association between vit D status and physical activity in the Prospective Epidemiological Research Studies in IRAN (PERSIAN) cohort study, while also considering the influence of potential confounders. This cross-sectional study has been conducted on ۹۵۲۲ participants aged ۳۵ to ۷۰. The serum vit D concentrations were measured, and participants were divided into three tertiles of low, moderate, and high levels of physical activity according to the metabolic equivalent of task (MET). The cutoff level for vit D deficiency was considered ≤۱۲ ng/ml. The association of vitamin D deficiency with physical activity (PA) was examined using logistic regression analysis. Odds ratio (OR)s were adjusted for vit D supplement use, sociodemographic characteristics, and additional covariates. About ۴۴۲۸ participants were male, and the mean age of participants was ۵۱.۴۶ ± ۸.۹۱ years. The prevalence of vit D deficiency was ۲۲.۸% and more common in women than men (۲۳.۸% vs. ۲۱.۷%). The prevalence of vit D deficiency decreased with increasing physical activity (P<۰.۰۰۱). In the adjusted model for vit D supplements and sociodemographic characteristics, the participants with low physical activity had a higher risk of vit D deficiency than participants with high physical activity. According to our findings, vit D deficiency was associated with a reduced frequency of participation in all forms of physical activity.A surprisingly high prevalence of vitamin D (vit D) deficiency has recently been reported worldwide. This study investigated the association between vit D status and physical activity in the Prospective Epidemiological Research Studies in IRAN (PERSIAN) cohort study, while also considering the influence of potential confounders. This cross-sectional study has been conducted on ۹۵۲۲ participants aged ۳۵ to ۷۰. The serum vit D concentrations were measured, and participants were divided into three tertiles of low, moderate, and high levels of physical activity according to the metabolic equivalent of task (MET). The cutoff level for vit D deficiency was considered ≤۱۲ ng/ml. The association of vitamin D deficiency with physical activity (PA) was examined using logistic regression analysis. Odds ratio (OR)s were adjusted for vit D supplement use, sociodemographic characteristics, and additional covariates. About ۴۴۲۸ participants were male, and the mean age of participants was ۵۱.۴۶ ± ۸.۹۱ years. The prevalence of vit D deficiency was ۲۲.۸% and more common in women than men (۲۳.۸% vs. ۲۱.۷%). The prevalence of vit D deficiency decreased with increasing physical activity (P<۰.۰۰۱). In the adjusted model for vit D supplements and sociodemographic characteristics, the participants with low physical activity had a higher risk of vit D deficiency than participants with high physical activity. According to our findings, vit D deficiency was associated with a reduced frequency of participation in all forms of physical activity.