Folic acid deficiency induces congenital malformations. Dihydrofolate reductase (DHFR) plays key roles in folic acid metabolism. The dysfunction of DHFR results in the inhibiting of folic acid. In vertebrate, pharyngeal arches are the progenitor of craniofacial structure and cardiac out flow tract. By using zebrafish as the animal model and coupling with gene knock-down and over-expression technologies, the role of dihydrofolate reductase gene (DHFR) in the development of pharyngeal arches were explored. In DHFR knock-down embryos, the malformations of pharyngeal arches and palates were showed by paraffin section and alcian blue staining. DHFR over-expressing can rescue these malformations. TBX1 and HAND2 are two key transcription factors in the development of pharyngeal arches. Whole-mount in situ hybridization and Real-time PCR were performed to detect the expression levels of TBX1 and HAND2. The expressions of TBX1 and HAND2 were reduced in DHFR knock-down group. DHFR over-expression can increase expressions of TBX1 and HAND2. These results demonstrated that DHFR was essential for the development of pharyngeal arches and DHFR regulated the development of pharyngeal arches by effecting the expressions of TBX1 and HAND2.