Although low vitamin D status resulting from night work is a suspected cause of various health disorders, few studies have investigated the association between night-shift work and vitamin D status. Here, we examined serum 25-hydroxyvitamin D (25OHD) levels in 19 Japanese indoor workers, including night-shift workers, in blood samples collected at the annual medical checkup (late July) in a metal tool factory. Analyses were finally restricted to 14 male workers (33-59 yr) in 3 groups: fixed daytime work (n=6), and rotating shift work with (n=4) and without (n=4) night shifts. No significant differences in serum 25OHD levels were observed among the three groups (p=0.98, Kruskal-Wallis test). One to two participants in each group had 25OHD levels lower than the 20 ng/ml reference value for vitamin D deficiency even in summer. These results clearly indicate the need for large-scale studies to test the hypothesis that night-shift work is associated with lower 25OHD levels.