The ventricular septal defect (VSD) is the most common congenital heart defect and no candidate susceptibility gene has been identified. Endocardial cushion and outflow septal morphogenesis, malalignment of which induces VSD, have been suggested to be mediated by the vascular endothelial growth factor (VEGF). Three single-nucleotide polymorphism (SNP) variants in promoter and 5'-UTR region of the VEGF gene, C-2578A (rs699947), G-1154A (rs1570360) and G-634C (rs2010963), were reported to alter its expression. We assessed the association in a Chinese population between these SNPs and VSD using a double approach: case-control and TDT designs. Among the three SNPs, only -634C allele was less frequently present in 222 patients compared to 352 controls (odds ratio: 0.76, 95% CI: 0.59-0.97, X(2)=5.06, P=0.024, not significant after a Bonferroni correction). This was significantly less transmitted to VSD patients (trios: 142) (odds ratio: 0.39, 95% CI: 0.25-0.62, X(2)=8.11, df=1, P=0.004, corrected P=0.024). A similar result was observed for haplotype -2578C/-1154G/-634C allele in both studies (in TDT: X(2)=7.51, df=1, P=0.006, corrected P=0.048). All these associations for the first time demonstrated that -634C allele was in a significant protective association against VSD, suggesting that VEGF dysregulation was involved in the pathological processes of VSD.