Here, we aimed to examine the associations between levels of serum 25-hydroxyvitamin D [25(OH)D] and inflammatory cytokines in healthy Japanese adults. A total of 95 healthy adults (61 women; age range 21-69 years) participated in our study. Fasting blood samples were analyzed for 25(OH)D, 1,25-dihydroxyvitamin D [1,25(OH)2D], interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-17 (IL-17) levels using enzyme-linked immunosorbent assays kits. Total percent body fat was determined by dual energy X-ray absorptiometry (DXA). Moderate to vigorous physical activity (MVPA) was assessed objectively using an activity monitor for 7 days. The mean 25(OH)D concentration was 34.7 nmol/L, and 83 subjects had 25(OH)D concentrations less than 50 nmol/L. Multiple linear regression analysis revealed that serum 25(OH)D level was positively related to plasma IL-17 level (β=0.26, p=0.025), after adjustment for gender, age, vitamin D intake, alcohol consumption, smoking status, and percent body fat. This relationship remained statistically significant (β=0.28, p=0.019) even after additional adjustment for MVPA. However, no significant association was found between serum 25(OH)D level and plasma IFN-γ or IL-6 levels. In conclusion, this study identified a high prevalence of vitamin D deficiency in healthy Japanese adults. Serum 25(OH)D level was positively related to IL-17 level, independent of physical activity.