MicroRNA-22 (miR-22) is emerging as a critical regulator in organ development and various cancers. However, its role in normal hematopoiesis and leukaemogenesis remains unclear. Here, we detected its increased expression during monocyte/macrophage differentiation of HL-60, THP1 cells and CD34+ hematopoietic stem/progenitor cells, and confirmed that PU.1, a key transcriptional factor for monocyte/macrophage differentiation, is responsible for transcriptional activation of miR-22 during the differentiation. By gain- and loss-of-function experiments, we demonstrated that miR-22 promoted monocyte/macrophage differentiation, and MECOM (EVI1) mRNA is a direct target of miR-22 and MECOM (EVI1) functions as a negative regulator in the differentiation. The miR-22-mediated MECOM degradation increased c-Jun but decreased GATA2 expression, which results in increased interaction between c-Jun and PU.1 via increasing c-Jun levels and relief of MECOM- and GATA2-mediated interference in the interaction, and thus promoting monocyte/macrophage differentiation. We also observed significantly down-regulation of PU.1 and miR-22 as well as significantly up-regulation of MECOM in acute myeloid leukemia (AML) patients. Reintroduction of miR-22 relieved the differentiation blockage and inhibited the growth of bone marrow blasts of AML patients. Our results revealed new function and mechanism of miR-22 in normal hematopoiesis and AML development and demonstrated its potential value in AML diagnosis and therapy.