Expression and activity of inwardly rectifying potassium (KIR) channels within the heart are strictly regulated. KIR channels have an important role in shaping cardiac action potentials, having a limited conductance at depolarized potentials but contributing to the final stage of repolarization and resting membrane stability. Impaired KIR2.1 function causes Andersen-Tawil Syndrome (ATS) and is associated with heart failure. Restoring KIR2.1 function by agonists of KIR2.1 (AgoKirs) would be beneficial. The class 1c antiarrhythmic drug propafenone is identified as an AgoKir; however, its long-term effects on KIR2.1 protein expression, subcellular localization, and function are unknown. Propafenone’s long-term effect on KIR2.1 expression and its underlying mechanisms in vitro were investigated. KIR2.1-carried currents were measured by single-cell patch-clamp electrophysiology. KIR2.1 protein expression levels were determined by Western blot analysis, whereas conventional immunofluorescence and advanced live-imaging microscopy were used to assess the subcellular localization of KIR2.1 proteins. Acute propafenone treatment at low concentrations supports the ability of propafenone to function as an AgoKir without disturbing KIR2.1 protein handling. Chronic propafenone treatment (at 25–100 times higher concentrations than in the acute treatment) increases KIR2.1 protein expression and KIR2.1 current densities in vitro, which are potentially associated with pre-lysosomal trafficking inhibition.