Abstract Objectives The inhibition of the cardiac rapid delayed rectifier potassium current (IKr) and its cloned equivalent human ether-a-go-go-related gene (hERG) channel illustrate QT interval prolonging effects of a wide range of clinically used drugs. In this study, the direct interaction of the intravenous anaesthetic ketamine with wild-type (WT) and mutation hERG currents (IhERG) was investigated. Methods The hERG channel (WT, Y652A and F656A) was expressed in Xenopus oocytes and studied using standard two-microelectrode voltage-clamp techniques. Key findings WT hERG is blocked in a concentration-dependent manner with IC50 = 12.05 ± 1.38 μm by ketamine, and the steady-state inactivation curves are shifted to more negative potentials (about −27 mV). The mutation to Ala of Y652 and F656 located on the S6 domain attenuate IhERG blockade by ketamine, and produced approximately 9-fold and 2.5-fold increases in IC50 compared with that of WT hERG channel, respectively. Conclusions Ketamine blocks WT IhERG expressed in Xenopus oocytes in a concentration-dependent manner and predominantly interacts with the open hERG channels. The interaction of ketamine with hERG channel may involve the aromatic residues Tyr652 and Phe656.