Introduction GFA (Guan-Fu Base A) as one of the main active substances in the Chinese medicine Ranunculaceae Aconite, has been approved the effect of anti-atrial fibrillation via its atrial-selective Na ⁺ channel-blocking action. It is recently undergoing phase IV clinical study. However, the molecular mechanism of Na _V1.5 channel inhibition by GFA is largely unclear. Methods and Results Na _V1.5 channel and its mutants were expressed in Xenopus oocytes and the currents were recorded with two-microelectrode voltage-clamp. GFA inhibited Na _V1.5 currents in a concentration-dependent manner, with IC ₅₀ of 66.24 μM, 371.59 μM, and 381.08 μM for wild type (WT), Delta KPQ (∆KPQ) and R1623Q constructs, respectively. Both the mutations of ∆KPQ and R1623Q decreased inhibitory potency of GFA about 5~6-fold. N406K mutation significantly altered the inhibition effect of GFA. Even 1 mM GFA has almost no inhibitory effect on the mutant. For both the WT and mutant channels, GFA reduced the currents in concentration, voltage and time dependent manner. Conclusion: GFA is a potent blocker of Na _V1.5 channel. N406, the aromatic residues in the transmembrane helical of DIS6, is most likely responsible for the high-affinity binding of GFA to Na _V1.5 channel.