The objective of this study was to examine whether high serum 25-hydroxyvitamin D (25(OH)D) concentration was associated with high skeletal muscle mass, taking into account the effects of sex and age among the participants of the Korea National Health and Nutrition Examination Survey (KNHANES) aged 40 years or older. This was a cross-sectional study using data from the 2009 to 2010 KNHANES; a total of 8406 subjects (3671 men and 4735 women) were included. The appendicular skeletal muscle mass index (ASMMI, kg/m2) was estimated to measure the skeletal muscle mass. Hypovitaminosis was classified when the level of serum 25(OH)D was <20 ng/ml. The general linear model adjusted for confounding factors was used to determine differences in means of ASMMI by 25(OH)D status. The mean values of ASMMI were higher for men when compared with women. Women had a greater proportion of hypovitaminosis (71·1 %) compared with men (53·2 %). After adjusting for multiple factors, men were seen to have significant differences in ASMMI based on 25(OH)D status regardless of age, showing a lower mean value of ASSMI in those with hypovitaminosis. However, there was no difference in ASMMI by 25(OH)D status among women in both younger and older age groups. In conclusion, we found that there might be a positive relationship between 25(OH)D and skeletal muscle mass in men, indicating that interventions to improve 25(OH)D levels that are aimed at increasing muscle mass could be beneficial for men with more rapid decreased rate of skeletal muscle mass.