NK cells are indispensable components of tissue microenvironment and play vital in both innate and adaptive immunity. The activation and function of NK cells are affected by tumor microenvironments. NK cells are also important players in leukemic microenvironment. However, their characteristics in leukemic microenvironment, including maturation status, phenotype, subpopulations and functional roles especially immunoregulatory potential, have not been well established. Here, we studied these characteristics of NK cells in MLL-AF9 induced mouse acute myeloid leukemia (AML) model. Increase of more mature NK cells were detected in the AML spleen. Splenic AML microenvironment promoted NK cell activation in early and middle stages of leukemia. Cytotoxicity molecules and cytokines were up-regulated in activated NK cells. Furthermore, NK cells from AML microenvironment regulated T cell function, not only by maintaining the activation of CD4+ and promoting the degranulation of cytotoxic CD8+ T cells but also by influencing the differentiation of CD4+ T cells. Moreover, two NK cell subpopulations marked by DNAM-1 (CD226) had distinct cytokine expression patterns but similar regulatory effects on T cells. Collectively, these findings highlight the significance of immunoregulatory role of NK cells, and suggest novel therapeutic potential for leukemia by manipulating NK cell immunoregulatory activity.Keywords: DNAM-1 (CD226); Immunoregulatory potential; Leukemic microenvironment; NK cells; T cells.