Female gender is a risk factor for drug-induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether-a-go-go related gene (hERG) channel. Some clinical evidence suggests that estrogen is a determinant of the gender-differences in drug-induced QT prolongation and baseline QT_C intervals. Although the chronic effects of estrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through estrogen receptors. We therefore investigated acute effects of the most bioactive estrogen, 17-estradiol (E2) at its physiological concentrations on cardiac repolarization and drug-sensitivity of the hERG (I_Kr) channel in Langendorff-perfused guinea pig hearts, patch-clamped guinea pig cardiomyocytes and culture cells over-expressing hERG. We found that physiological concentrations of E2 partially suppressed I_Kr in a receptor-independent manner. E2-induced modification of voltage-dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug-binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QT_C prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031-sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031-induced QT_C prolongation, and may provide a novel mechanism for the higher susceptibility to drug-induced arrhythmia in women.