Acute myeloid leukemia (AML) is a heterogeneous hematopoietic disorder with a poor prognosis. Abnormal DNA methylation is involved in the initiation and progression of AML. The de novo methyltransferases Dnmt3a and Dnmt3b are responsible for the generation of genomic methylation patterns. While DNMT3A is frequently mutated in hematological malignancies, DNMT3B is rarely mutated. Although it has been previously reported that Dnmt3b functions as a tumor suppressor in a mouse model of Myc-induced lymphomagenesis, its function in AML is yet to be determined. In this study, we demonstrated that deletion of Dnmt3b accelerated the progression of MLL-AF9 leukemia by increasing stemness and enhancing cell cycle progression. Gene profiling analysis revealed upregulation of the oncogenic gene set and downregulation of the cell differentiation gene set. Furthermore, loss of Dnmt3b was able to synergize with Dnmt3a deficiency in leukemia development. Taken together, these results demonstrate that Dnmt3b plays a tumor suppressive role in MLL-AF9 AML progression, thereby providing new insights into the roles of DNA methylation in leukemia development.