BACKGROUND: Spina bifida is a class of neural tube defects, which are congenital malformations of the central nervous system with a prevalence of 0.5 to 12 per 1000 births globally. In this article we attempt to identify genes related to folate and its metabolic pathways that are involved in the etiology of spina bifida. METHODS: We selected 50 folate metabolism-related genes and genotyped polymorphisms in those genes. Eighty-seven polymorphisms in 45 genes passed quality controls. Associations with spina bifida were investigated in 180 patients and 190 controls. For those polymorphisms that were nominally associated with spina bifida risk, the relation with serum and red blood cell folate, vitamin B(12), and homocysteine was evaluated in controls. RESULTS: A polymorphism in CUBN was significantly associated with decreased spina bifida risk, after correction for multiple testing, and was related to increased vitamin B(12) (p = 0.039) and red blood cell folate (p = 0.001). The CUBN gene encodes the intrinsic factor-cobalamin receptor (or cubilin), a peripheral membrane protein that acts as a receptor for intrinsic factor-vitamin B(12) complexes. Vitamin B(12) is an important cofactor in the folate metabolism, and low B(12) status in mothers has been linked to neural tube defects in children. Other interesting findings include nominally significant associations with polymorphisms in TRDMT1, ALDH1L1, SARDH, and SLCA19A1 (RFC1). CONCLUSION: Our study indicates interesting new candidate genes and functional pathways for further study and confirms earlier findings. None of the genes CUBN, TRDMT1, ALDH1L1, or SARDH have been investigated previously for association with spina bifida.