Cleft lip and cleft palate are common congenital craniofacial birth defects in humans. Phenytoin (PHT) is a risk factor of cleft palate formation; however, the molecular mechanisms by which phenytoin exerts its teratogenic effects resulting in cleft palate remain unknown. The Satb2 gene mutation is associated with cleft palate. Satb2-deficient mice exhibit cleft palate deformity and an up-regulation of Hoxa2 in the fronto-nasal region. In this study, phenytoin was administered intraperitoneally to pregnant C57BL/6 mice on the 10th day of gestation. Real-time PCR results showed that the expressions of Satb2 and Hoxa2 in craniofacial tissues of mouse embryos were obviously different at different time points. The Satb2 gene was down-regulated and the Hoxa2 gene was up-regulated in phenytoin-treated mouse embryonic craniofacial tissue. We conclude that phenytoin may regulate the expression of these two genes in C57BL/6 mice and it may also be involved in the formation of cleft palate.