Ranolazine, an anti-ischemic agent, inhibits IKr [encoded by the human ether-a-go-go-related gene (HERG)] and causes a small QT interval prolongation without any proarrhythmic events. The objective of this study was to elucidate the biophysical characteristics of inhibition of HERG K+ current (IHERG) by ranolazine. We investigated the effects of ranolazine using voltage-clamp and Western blot analyses of HERG channels stably expressed in HEK293 cells. Ranolazine reduced IHERG with the half-maximal inhibitory concentration of 12.0 μM. Block of IHERG by ranolazine was reversible and voltage-dependent but frequency-independent. At 0 mV, the time constants for development of block were 76.6 ± 1.6, 35.8 ± 2.4, and 19.4 ± 1.7 msec with 10, 30, and 100 μM ranolazine (n = 4), respectively. The apparent dissociation constant estimated from the time course of ranolazine-induced IHERG decay was 22.5 μM. After repolarization at −80 and −100 mV, IHERG recovery from ranolazine block followed a monophasic time course with τ values of 204.3 ± 51.5 and 155.0 ± 31.9 msec (n = 5), respectively. Intracellular but not extracellular application of a membrane-impermeable (permanently charged) ranolazine analogue caused rapid block of IHERG. Ranolazine did not alter HERG protein trafficking to the plasma membrane. In conclusion, ranolazine caused a time- and voltage-dependent, but frequency-independent, block of IHERG. The kinetics of IHERG inhibition (at positive potentials) and unblock (upon hyperpolarization) by ranolazine were rapid. These distinct and rapid kinetic interactions of ranolazine with IHERG may partially contribute to the observations that the drug is not proarrhythmic despite causing a small prolongation of action potentials and QT intervals.