Previous studies have demonstrated that male hypogonadism is associated with a low level of vitamin D. However, no reports have investigated the effects of vitamin D on testosterone levels in Korean men. Our aim was to investigate whether testosterone levels are associated with serum vitamin D levels and whether seasonal variation exists. This cross-sectional study analyzed serum 25-hydroxyvitamin D [25(OH)D], total testosterone (TT), and free testosterone (FT) in 652 Korean men over 40 years of age who had undergone a comprehensive medical examination. The average age of the subjects was 56.7 ± 7.9 years, and the mean serum 25(OH)D, TT and FT levels were 21.23 ± 7.9 ng ml-1 , 4.70 ± 1.6 ng ml-1 , and 8.12 ± 3.3 pg ml-1 , respectively. In the multiple linear regression model, 25(OH)D showed positive association with TT (β =0.137, P< 0.001) and FT (β =0.103, P= 0.008). 25(OH)D and FT showed similar seasonal or monthly variation after adjustment for age. A vitamin D deficiency [25(OH)D < 20 ng ml-1 ] was associated with an increased risk of deficiencies of TT (<2.30 ng ml-1 ) (odds ratio [OR]: 2.65; 95% confidence interval [CI]: 1.21-5.78, P= 0.014) and FT (<6.50 pg ml-1 ) (OR: 1.44; 95% CI: 1.01-2.06 P= 0.048) after adjusting for age, season, body mass index, body composition, chronic disease, smoking, and alcohol use. In conclusion, we demonstrated a positive correlation between 25(OH)D and testosterone, which showed similar seasonal variation in Korean men.