Epidemiological studies have shown that the risks of fetal malformation such as neural tube defects increase in diabetic pregnancy. To explore the mechanism of fetal malformation induced by diabetes, cDNA subtraction using mouse embryos (E9.5) of diabetic dams and those of controls was performed to identify differentially expressed genes. The expression level of genes identified by cDNA subtraction was further verified by quantitative RT-PCR using E8.5 embryos, and differential expression of 4 genes, Brcc3, Commd3, Ddx1, and SET was confirmed. We also analyzed the expression level of neural tube defect-related genes, and found that Folbp1, EphrinA5 and Sox10 were differentially expressed. Altered expression of these genes mostly persisted throughout the later stages of the development (E10.5-14.5). Hierarchical clustering analysis showed correlation between expression levels of these genes, suggesting that these genes cooperatively play a role in embryonic development. Our results suggest that an altered gene expression profile in embryos underlies the development of congenital malformation in diabetic pregnancies.