Current guidelines provide a universal recommendation on vitamin D intake to prevent insufficiency. However, the relative influence of food, UVB and other factors on serum 25-hydroxyvitamin D (25(OH)D) insufficiency has been poorly investigated in preschool children. We assessed serum 25(OH)D quantities and their association with vitamin D intake using a brief-type self-administered diet history questionnaire for children aged 3–6 years (BDHQ3y), outdoor playing time and background UVB radiation level among 574 36-month-old Japanese children living at latitude 35°N. The average serum 25(OH)D concentration was 23·5 (sd 6·1) ng/ml, and 170 (29·6 %) children had vitamin D insufficiency (<20 ng/ml) despite high consumption of fish. Multiple logistic regression adjusting for social factors showed that when background UVB radiation level was <15 kJ/m2 (monthly average), there was a 1·89 (95 % CI 1·31, 2·74) times higher risk of vitamin D insufficiency, to which vitamin D intake nor time spent outdoors were significantly associated. ANOVA showed that the contribution of the variability in vitamin D intake on the variability of serum 25(OH)D level was 1·8 % of that of UVB exposure. The correlation between vitamin D intake and serum 25(OH)D level was not stronger when limited to measurements in winter. We found that nearly 30 % of 3-year-old Japanese children had vitamin D insufficiency despite high consumption of fish and living at relatively low latitude. We failed to observe an association between vitamin D intake and the risk of vitamin D insufficiency. This may be due to the extremely limited access to vitamin D-fortified food and supplements for children in Japan.