BACKGROUND: Although several studies have suggested a potential positive association between serum vitamin D levels and muscle strength in older adults, it remains unclear whether this relationship is sex-dependent. AIM: To evaluate the association between serum 25 hydroxyvitamin D [25(OH)D] levels and muscle strength in older adults and to assess whether this association is dependent on sex. METHODS: This cross-sectional study utilized data from the National Health and Nutrition Examination Survey (NHANES) conducted between 2001 and 2002. A total of 1173 individuals aged 50-85 years (630 men and 543 women) were included in the analysis. The peak isokinetic force of the knee extensor was assessed using a kinetic communicator isokinetic dynamometer. Serum 25(OH)D levels were measured using the standardized liquid chromatography-tandem mass spectrometry method. Linear regression analyses were conducted to estimate coefficients and 95% confidence intervals for muscle strength across tertiles of serum vitamin D levels, adjusting for potential confounders. RESULTS: Tertiles of serum vitamin D were positively associated with muscle strength in older men (p-trend = 0.007), whereas no association was observed in older women (p-trend = 0.140) or in the total sample (men and women together; p-trend = 0.139). CONCLUSION: Serum vitamin D levels were positively associated with muscle strength specifically in older men, whereas no association was observed in older women. These results suggest that the relationship between vitamin D and strength may be sex-dependent.