Objectives Iloperidone is an atypical antipsychotic drug that is widely used for the treatment of schizophrenia. hERG 3.1, alternatively spliced form of hERG 1A, is considered a potential target for an antipsychotic drug. The present study was designed to study the effects of iloperidone on hERG 1A/3.1 heterotetrameric channels. Methods The interactions of iloperidone with hERG 1A/3.1 heterotetrameric channels stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and western blot analysis. Results Iloperidone inhibited the hERG 1A/3.1 tail currents at −50 mV in a concentration-dependent manner with an IC50 value of 0.44 μM. The block of hERG 1A/3.1 currents by iloperidone was voltage-dependent and increased over a range of voltage for channel activation. However, the block by iloperidone was voltage-independent at more depolarized potentials where the channels were fully activated. A fast application of iloperidone inhibited the hERG 1A/3.1 current elicited by a 5-s depolarizing pulse to +60 mV to fully inactivate the hERG 1A/3.1 currents. Iloperidone also induced a rapid and reversible inhibition of hERG 1A/3.1 tail currents during repolarization. However, iloperidone had no effect on either hERG 1A or hERG 1A/3.1 channel trafficking to the cell membrane. Conclusions Our results indicated that iloperidone concentration-dependently inhibited hERG 1A/3.1 currents by preferentially interacting with the open states of channels, but not by the disruption of membrane trafficking or surface membrane expression of hERG 1A and hERG 1A/3.1 channel proteins.