Acute myeloid leukemia (AML) is a type of hematologic diseases, which is related to abnormal genes. The aberrant microtubule actin cross-linking factor 1 (MACF1) is associated with progression of multiple tumors by initiating cell proliferation. Nevertheless, the function and action mechanism of MACF1 in AML cell proliferation remain mostly unknown. Our study aimed to explore the influence of MACF1 on AML cell proliferation by CCK-8 and EdU staining assays. Moreover, we aimed to explore the effect of MACF1 on downstream Runx2 and the PI3K/Akt signaling. MACF1 expression in AML patients was predicted by bioinformatics analysis. Cells were transfected with si-con, si-MACF1 or Runx2 using Lipofectamine 2000. Upregulated MACF1 was found in AML patients and predicted worse overall survival. MACF1 expression was upregulated in AML cells compared with that in hematopoietic stem and progenitor cells. MACF1 silencing reduced AML cell proliferation. Runx2 level was increased in AML cells, and decreased by silencing MACF1. Runx2 upregulation rescued MACF1 silencing-mediated inhibition of proliferation. MACF1 downregulation inhibited activation of the PI3K/Akt pathway by decreasing Runx2. Activation of the PI3K/Akt pathway abrogated the suppressive role of MACF1 downregulation in AML cell proliferation. In conclusion, MACF1 knockdown decreased AML cell proliferation by reducing Runx2 and inactivating the PI3K/Akt signaling.