The article evaluated the inhibitory action of procaine on wild-type and mutated HERG potassium channel current (I_HERG) to determine whether mutations in the S6 region are important for the inhibition of I_HERG by procaine. HERG channels (WT, Y652A, and F656A) were expressed in Xenopus laevis oocytes and studied using the standard two-microelectrode voltage-clamp technique. The results revealed that WT HERG is blocked in a concentration-, voltage-, and state-dependent manner by procaine ([IC₅₀] = 34.79 μM). The steady state activation curves slightly move to the negative, while inactivation parameters move to the positive in the presence of procaine. Time-dependent test reveals that voltage-dependent I_HERG blockade occurs extremely rapidly. Furthermore, the mutation to Ala of Y652 and F656 produce about 11-fold and 18-fold increases in IC₅₀ for I_HERG blockade, respectively. Simultaneously, for Y652A, the steady state activation and inactivation parameters are shifted to more positive values after perfusion of procaine. Conclusively, procaine state-dependently inhibits HERG channels (WT, Y652A, and F656A). The helix residues Y652 and F656 in the S6 transmembrane domain might play a role in interaction of the drug with the channel.