Azelastine is a second gengeration histamine H1 receptor antagonist used as an anti-asthmatic and anti-allergic drug that can induce QT prolongation, which may lead to torsades de pointes. 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 azelastine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Azelastine increased the action potential duration at 90% of repolarization (APD90) in a concentration-dependent manner, with an IC50 of 1.08 μΜ when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of azelastine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Azelastine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 for the azelastine-induced block of the hERG currents in HEK293 cells at 36 °C was 11.43 μΜ at +20 mV, while the drug blocked L-type Ca2+ channel expressed in HEK293 cells with an IC50 of 26.21 μΜ. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that azelastine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of azelastine.