The regulator of G-protein signalling (RGS) proteins are a family of well-known GTPase-activating proteins that negatively regulate G-protein cycle. Cardiac predominant subtype, RGS4, plays an important role in modulating the M2 muscarinic receptor (M2R)-activated G-protein-gated inwardly rectifying K⁺ (K_G) currents. However, the mechanism of RGS4-mediated regulation still remains unclear. By investigating K_G currents in rat atrial myocytes and Xenopus oocyte expression system, I found that RGS4 is a determinant for partial agonism of M2R. M2R partial agonist pilocarpine acted like a full agonist in the absence of RGS4. RGS4 suppressed the pilocarpine-evoked K_G currents in a pilocarpine concentration- and voltage-dependent manner. Hyperpolarization enhanced the RGS4-mediated regulation of K_G. These data help us to understand the molecular components and mechanism underlying the RGS4-mediated regulation on the M2R-activated physiological responses.