Inhibition of K⁺-conductance through the hERG channel leads QT prolongation and associates with cardiac arrhythmias. We have found that estrogens interact with a drug-binding site, F656 of the hERG channel, and alter effects of a hERG blocker. However precise mechanistic insights have not been elucidated. We here investigated actions of ethynylestradiol (EE2), a synthetic estrogen, on the hERG channel. HEK293 cells stably-expressing the hERG channels were cultured in the steroid-free medium. The patch-clamp technique is performed to record hERG currents. Supratherapeutic concentrations of EE2 did not alter amplitudes and kinetics of the hERG currents elicited with train pulses at 20 mV (0.1 Hz). On the other hand, EE2 recovered the hERG inhibition induced by E-4031. These results suggest that EE2 interacts with E-4031 at the promiscuous drug-binding site of the hERG channel and imply that EE2, an anti-breast cancer drug or an oral contraceptive, is protective against drug-induced QT prolongation.