In order to clarify the effects of 17beta-estradiol (E2) on natural killer (NK) cells and the possibly regulatory mechanisms, we obtained highly purified and viable NK cells from C57BL/6J mouse spleen by a magnetic cell sorter (MACS). These cells were treated with E2 and then their cytotoxicity and proliferative capacity were examined. To further investigate the mechanisms on the effect of E2 on NK cells, expressions of activation-associated markers (CD69, CD122) and inhibitory receptors (CD94, Ly49), and intracellular cytokine production were analyzed. At last, we performed the cDNA microarray to explore the possible involved genes. We found that E2 could suppress NK cell cytotoxicity and proliferative capacity in vitro. E2 reduced NK cell cytotoxicity and proliferative capacity, which may be through influencing the phenotypes and cytokine expression of NK cells, mainly involving CD94 and IFN-gamma. Furthermore, regulation of Stat4, Fyn, Sh2d1a, Eat2, Cd244, Irf1, Runx1, Irf7, Irf5, Esrra and Nr5a1 genes may be related to the cytotoxicity, proliferation and cytokine production of E2-mediated purified NK cells.