Methotrexate (MTX), an antagonist of folic acid, can inhibit dihydrofolate reductase (DHFR) which is of great importance in the synthesis of tetrahydrofolic acid and embryonic development. In this study, we found that after being exposed to 1.5 mM MTX at 6-10 hours post-fertilization, zebrafish embryos fail to form normal cardiovascular system. In MTX-treated embryos, the morphological development of ventricle and atrium was disrupted, the cardiac twist was abnormal, the heart rate and ventricular shortening fraction were reduced, and the vascular development was disrupted. We also found that either microinjection with dhfr-gfp mRNA or treatment with folinic acid calcium salt pentahydrate (CF) could cause improved development in the heart and vessels in MTX-treated embryos, which proved that MTX induced the malformations by inhibiting DHFR. The transcript levels of genes such as hand2, mef2a, mef2c, and flk-1 were reduced in MTXtreated embryos. Compared with the MTX-treated group, the transcript levels of hand2, mef2a, mef2c, and flk-1 were increased in the MTX 1 dhfr-gfp mRNA injected group and in the MTX 1 CF group. Our results indicated that the disrupted development of the heart and vessels in MTX-treated embryos is related to the reduced transcript levels of hand2, mef2a, mef2c, and flk-1.