Summary Most Japanese pregnant women do not take the estimated average requirement (EAR) of folate for pregnant women, which is 400 mg/d. Nevertheless, folate deficiencies have not been reported. We examined biomarkers for evaluating the status of folate in pregnant Japanese women. Apparently healthy pregnant Japanese women were cross-sectionally recruited from a private obstetric hospital. We measured nutritional biomarkers of folate in these women, as well as their folate intake. The numbers of subjects were 230 (49, 62, and 81, and 38 in the first (up to 15 wk), second (16-30 wk), and third (over 31 wk) trimesters of pregnancy, and 1 mo after delivery, respectively). Folate intakes (medians) in the first, second, and third trimesters, and 1 mo after delivery were 2356147 (194), 226683 (218), and 256685 (254), and 3006105 (305) mg/d, respectively. Folate concentrations in plasma and erythrocytes appeared to be valid indicators for assessing folate status, with cutoff values of 7 nmol/L and 300 nmol/L, respectively. Plasma folate concentrations (medians) in the first, second, and third trimesters, and 1 mo after delivery were 17.669.6 (16.7), 12.468.3 (9.4), and 12.168.4 (9.4), and 10.768.9 (7.9) nmol/L, respectively. Each of the folate values was over the cut-off value. Erythrocyte folate concentrations (medians) in the first, second, and third trimesters, and 1 mo after delivery were 3586108 (365), 3896154 (365), and 3256150 (315), and 3086158 (276) nmol/L, respectively. Each of the folate values was near or over the cut-off value. In conclusion, the data obtained demonstrated that the estimated average requirement of folate for pregnant Japanese women was 250 mg/d.