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Background: Pilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. Methods: HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments. Results: Acute exposure to pilsicainide at 0.03–10 μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03–10 μM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W. Conclusions: Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.