TXN2 encodes human thioredoxin 2, a small redox protein important in cellular antioxidant defenses, as well as in the regulation of apoptosis. Txn2 knockout mice fail to complete neural tube closure by E10.5 and die in utero. We hypothesized that genetic variation in human TXN2 gene may alter the function of the encoded protein in a manner associated with an increased risk for neural tube defects (NTDs). A DNA re-sequencing effort of the human TXN2 gene was taken. After a variation in the promoter was identified, the transcriptional activity of different alleles was investigated. The possible association between these variations and the risk of spina bifida was further evaluated in a subset of samples obtained from a large population-based case-control study in California in two different ethnicity groups, non-Hispanic white and Hispanic white. We identified a novel promoter insertion polymorphism located 9 base pairs upstream of the transcription start site of exon 1(-9 insertion). The GA, G and GGGA insertions were associated with a marked decrease of transcriptional activity when overexpressed in both U2-OS (an osteosarcoma cell line) and 293 cells (derived from human embryonic kidney). Further analysis revealed that the GA insertion was associated with increased spina bifida risk for Hispanic whites. Our study revealed a novel Ins/Del polymorphism in the human TXN2 gene proximal promoter region that altered the transcriptional activity and is associated with spina bifida risk. This polymorphism may be a genetic modifier of spina bifida risk in this California population. © 2009 Wiley-Liss, Inc.