This study examines the independent and combined effects of serum 25-hydroxyvitamin D (25(OH)D) levels and physical activity (PA) on phenotypic age (PhenoAge) in the U.S. population. Using data from the National Health and Nutrition Examination Survey (NHANES), the study found that higher 25(OH)D levels and adequate PA were associated with a lower risk of biological aging. The combined effect of high 25(OH)D and adequate PA was particularly significant in individuals aged 65 years or younger, with a 30.7% reduction in PhenoAgeAccel attributed to both factors. Combining 25(OH)D levels above 80.4 nmol/L with adequate PA reduced PhenoAge by 1.291 years. The study also found a multiplicative interaction between 25(OH)D and PA in individuals aged 65 years or younger, suggesting that the protective effects of both factors are synergistic. These findings highlight the importance of outdoor PA in slowing down the aging process and maintaining a healthy lifestyle. The study underscores the need for maintaining adequate vitamin D levels and regular physical activity to delay aging and reduce the risk of age-related diseases. The results emphasize the potential of combining vitamin D and PA to enhance protective effects, especially in middle-aged and younger adults. The study also notes the importance of further research to explore the mechanisms underlying these associations and to develop effective interventions for aging prevention.This study examines the independent and combined effects of serum 25-hydroxyvitamin D (25(OH)D) levels and physical activity (PA) on phenotypic age (PhenoAge) in the U.S. population. Using data from the National Health and Nutrition Examination Survey (NHANES), the study found that higher 25(OH)D levels and adequate PA were associated with a lower risk of biological aging. The combined effect of high 25(OH)D and adequate PA was particularly significant in individuals aged 65 years or younger, with a 30.7% reduction in PhenoAgeAccel attributed to both factors. Combining 25(OH)D levels above 80.4 nmol/L with adequate PA reduced PhenoAge by 1.291 years. The study also found a multiplicative interaction between 25(OH)D and PA in individuals aged 65 years or younger, suggesting that the protective effects of both factors are synergistic. These findings highlight the importance of outdoor PA in slowing down the aging process and maintaining a healthy lifestyle. The study underscores the need for maintaining adequate vitamin D levels and regular physical activity to delay aging and reduce the risk of age-related diseases. The results emphasize the potential of combining vitamin D and PA to enhance protective effects, especially in middle-aged and younger adults. The study also notes the importance of further research to explore the mechanisms underlying these associations and to develop effective interventions for aging prevention.