Hydroxyzine is a potent first-generation histamine H1 receptor antagonist that can increase action potential duration and induce QT prolongation in several animal models. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of leading causes of acquired long QT syndrome, we investigated the acute effects of hydroxyzine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of hydroxyzine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Hydroxyzine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 of hydroxyzine-dependent hERG block in Xenopus oocytes increased progressively relative to the degree of depolarization. Hydroxyzine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutation Y652A attenuated the hERG current block. The IC50 value of hydroxyzine-dependent hERG block increased from 5.9 μΜ to 35.1 μΜ by the mutation from tyrosine to alanine at 652 amino acid of the channel. These results suggest that the H1 antihistamine, hydroxyzine is a blocker of the hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.