Background: In recent years, a resurgence in the number of infants with vitamin D deficiency has been noted. In addition to seasonal differences in exposure to ultraviolet (UV) rays, regional differences in dietary habits and lifestyles may affect susceptibility to vitamin D deficiency. No studies have been conducted, however, on infants in multiple regions of Japan to determine the extent of differences in vitamin D status. Methods: 25-Hydroxyvitamin D (25OHD) was measured on radioimmunoassay in 126 infants aged 2-4 years, who participated in the Pilot Study of the Japan Environment and Children's Study (JECS) by the Ministry of Environment of Japan. A multiple regression model with 25OHD level as the outcome variable, and season and region as explanatory variables, was generated. Results: Both region and season during which infants participated in this study significantly affected 25OHD level (P = 0.0087 and <0.0001, respectively; Wald test). Reflecting decreased exposure to UV rays, infants who were examined in winter had lower 25OHD than those examined in summer. Infants from both Fukuoka Prefecture (33°N) and Kumamoto Prefecture (32°N), however, had lower 25OHD than those from Tochigi Prefecture (36°N), contrary to expectations given the extent of UV exposure. Conclusions: Regional differences in daily habits and/or environmental factors affect 25OHD level in Japanese infants. The JECS is expected to identify those factors to provide guidance on preventing infantile vitamin D deficiency.