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.