Aberrant expression of microRNA-92a (miR-92a) has been investigated in various cancers. However, the function and mechanism of miR-92a in acute myeloid leukemia (AML) remain to be elucidated. Our data showed that miR-92a was evidently downregulated and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was remarkably upregulated in AML cell lines HL-60 and THP-1. Dual luciferase reporter assay revealed that MTHFD2 was a direct target of miR-92a. Gain- and loss-of-function analysis demonstrated that MTHFD2 knockdown or miR-92a overexpression notably inhibited proliferation and promoted apoptosis of AML cell lines. Restoration of MTHFD2 expression reversed proliferation inhibition and apoptosis induction of AML cells triggered by miR-92a. Moreover, an implanted tumor model in mice indicated that miR-92a overexpression dramatically decreased tumor growth and MTHFD2 expression in vivo. Taken together, our results suggest that miR-92a inhibits proliferation and induces apoptosis by directly regulating MTHFD2 expression in AML. miR-92a may act as a tumor suppressor in AML, providing a promising therapeutic target for AML patients.