This study aimed to characterize serum 25-hydroxyvitamin D (25OH-D) values among Japanese children aged ≤48 mo. The study included 290 healthy infants and young children aged 0-48 mo (males/females=166/124) living in Shizuoka or Tokyo. The subjects were divided into three groups by age (Low Age: 0-5, Middle Age: 6-15, High Age: 16-48 mo). The vitamin D deficient state was defined as 25OH-D <12 ng/mL, the insufficient state as 12-20 ng/mL, and the sufficient state as >20 ng/mL. The seasonal variation of serum 25OH-D levels was also analyzed. The median serum 25OH-D levels in each group were: Low Age (n=50), 19 ng/mL; Middle Age (n=94), 30 ng/mL; and High Age (n=146), 30 ng/mL. The serum 25OH-D level was significantly lower in the Low Age group than in the other groups (p<0.01). Serum 25OH-D levels in summer and autumn (n=149) were significantly higher than in winter and spring (n=141) (33 vs. 25 ng/mL, p<0.01). In the Low Age group, there was a significant difference in serum 25OH-D levels between breast-fed infants (n=26) and formula-fed or mixed-fed infants (n=19) (12 vs. 32 ng/mL, p<0.01). However, there were no significant differences in 25OH-D levels between the two season classifications in either breast-fed or formula-fed and mixed-fed infants. Although clinical symptoms were not available, more than 75% of the breast-fed infants and 14.6% of infants and young children to whom food had been introduced were defined as having a vitamin D deficient or insufficient state. Breastfeeding seems one of the contributing factor to lower serum 25 OH-D levels among infants ≤5 mo of age.