GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata and inhibits atrial fibrillation potentiated by dilatation in heart by blocking the stretch-activated (SA) ion channels. However, the mechanism by which GsMTx-4 inhibits SA channels remains unknown. Here we report that the extracellularly applied nM concentrations of GsMTx4 inhibited SAKca channels in excised inside-out patch membranes of isolated chick heart cells. We found that GsMTx4 inhibited the stretch-induced SAKca channel opening in a dose-dependent manner without altering the single-channel conductance. Membrane stretch shifted the voltage dependence of activation of SAKca to more negative voltages, whereas a relatively low concentration of extracellularly applied GsMTx-4 could reverse this effect. Detailed single-channel kinetics analysis revealed that GsMTx-4 could antagonize the increases in the open-time duration induced by membrane stretch. In addition, we found that the sensitivity of SAKca channel towards GsMTx-4 effect was eliminated by the deletion of 59-amino-acid STREX (stress axis regulated exon) located between RCK1 and RCK2 domains in the C-terminus of SAKca channel, suggesting that STREX is the potential target site for GsMTx-4 action in SAKca channel. Our data establish that GsMTx-4 acts as a gating modifier on SAKca channel from chick heart, and may represent a common inhibition mechanism of GsMTx-4 against all types of mechanosensitive (MS) ion channels. Our finding also suggest SA channel is a potential target for curing atrial fibrillation by using GsMTx-4, thus may provide a new insight into the treatment of cardiac arrhythmia.