Objective：This study aims to investigate the effects of DNA methylation on germ layer differentiation of Xenopus laevis. Methods：Embryos were treated with 30 μmol/L 5-aza-2’-deoxycytidine（5-AZA-CdR）to inhibit DNA methyltransferase 1（DNMT1）in early embryonic cells，then observe the phenotype. Gastrula embryos were collected and the expression of three germ-layer marker genes was detected via in situ hybridization. To knock down endous DNMT1，the specific DNMT1 antisense oligonucleotide（DNMT1MO）were injected into 2-cell stage embryos，and then the expression of three germ-layer marker genes was detected by in situ hybridization. Results：5-AZA-CdR treatment led to abnormal development of embryos and inhibited the expression of the mesoderm marker gene Xbra. Knockdown of DNMT1 promotes expression of the dorsal mesodermal marker genes GSC and Chordin，and inhibits the expression of the ventral mesoderm marker gene Wnt8. Conclusion：DNA methyltransferase is involved in the regulation of the differentiation of the three germ layers of Xenopus laevis.