Objective To study the effects of estrogens (17α-ethynylestradiol and 17β-estradiol) on the ATP-sensitive potassium channel (K_ATP) activities in the enzymatically (collagenase and protease) isolated ICR mouse ventricular cardiomyocytes using inside-out and cell-attached patch clamp techniques. Methods In the inside-out patch configurations, the two estrogens reduced the K_ATP channel activities in a dose-dependent manner at -60 mV holding potential (HP). Results The half-maximal inhibitory concentration (IC50) of the 17α-esthynylestradiol and the 17β-estradiol were 0.3 μmol/L and 0.1 nmol/L, respectively. However, these agents had no effects on the pinacidil- and DNP-induced K_ATP channel activities in the cell-attached patch configuration. However, the inhibitory effect of K_ATP channel activities by the 17-estrogens or the anti-estrogens in the excised inside-out patch configuration were abolished by 1 pmol/L PDBu. Conclusions The results of this study demonstrate that some estrogens decrease the K_ATP channel activity acting on the inside of the isolated cardiomyocytes, and play a protective role to cardiomyocytes.