Abstract： Objective To evaluate the association between maternal serum levels of vitamin B12 and folate and neural tube defects (NTD) in Shanxi Province which with the extremely high prevalence of NTD in China. Methods A case-control study was conducted in five counties of Shanxi Province, China. Maternal concentrations of vitamin B12 and folate [expressed as G (P5-P95)]in NTD-affected pregnancies were dichotomized at a cut-off of 10th percentile of the mean values of control women to calculate the risk of NTD for the lower level group. Results The mean levels of serum vitamin B12 and folate in the NTD pregnancies were significantly lower than in the control [vitamin B12 83. 8 pmol/L(30. 5-209. 1) pmol/L vs 102. 6 pmol/L(51. 9-269. 7) pmol/L, P<0. 05; folate:10. 5 pmol/L(4. 4-24. 5) nmol/L vs 12. 9 pmol/L(6. 3-32. 7) nmol/L, P<0. 01]. In cases complicated with spina bifida, lower maternal serum vitamin B12 and folate levels were detected compared with the control group. While, in those complicated with anencephaly, only lower levels of maternal folate were shown. There was no statistical significance in maternal serum concentrations of vitamin B12 and folate between those cases with encephalocele and controls. Higher risk of NTDs were found in pregnant women with lower level of vitamin B12 and folate (AOR = 2. 58, 95% CI: 1. 20-5. 51; AOR= 2. 76, 95%CI: 1. 30-5. 87). Conclusions Deficiency or insufficiency of folate or vitamin B12 is associated with the increased risk of NTD, especially in high risk population.