Potassium (K(+)) channels have been implicated in proliferation of some tumor cells. However, whether K(+) channels are important to the pathogenesis of endometrial cancer (EC) remains unknown. In the present study, we report that intermediate-conductance Ca(2+)-activated K(+) (IKCa1) channels play a critical role in the development of EC. The expression of IKCa1 at both mRNA and protein levels in EC tissues was greatly increased than that in atypical hyperplasia and normal tissues. Treatment of EC cells with clotrimazole and TRAM-34, two agents known to inhibit IKCa1 channels, suppressed the proliferation of EC cells and blocked EC cell cycle at G(0)/G(1) phase. Similarly, downregulation of IKCa1 by siRNA against IKCa1 inhibited EC cell proliferation and arrested its cell cycle at G(0)/G(1) phase. A clotrimazole-sensitive K(+) current was induced in EC cells in response to the increased Ca(2+). The current density induced by Ca(2+) was greatly reduced by clotrimazole, TRAM-34, charybdotoxin or downregulation of IKCa1 by the siRNA against IKCa1. Furthermore, TRAM-34 and clotrimazole slowed the formation in nude mice of tumor generated by injection of EC cells. Our results suggest that increased activity of IKCa1 channel is necessary for the development of EC.