Tetrodotoxin (TTX) has been used for many decades to characterize the structure and function of biological ion channels. Yet, the precise mechanism by which TTX blocks voltage-gated sodium (Na-V) channels is not fully understood. Here molecular dynamics simulations are used to elucidate how Trx blocks mammalian voltage-gated sodium (Na-V) channels and why it fails to be effective for the bacterial sodium channel, Na(v)Ab. We find that, in Na(v)Ab, a sodium ion competes with TTX for the binding site at the extracellular end of the filter, thus reducing the blocking efficacy of TTX. Using a model of the skeletal muscle channel, Na(V)1.4, we show that the conduction properties of the channel observed experimentally are faithfully reproduced. We find that TTX occludes the entrance of Na(v)1.4 by forming a network of hydrogen-bonds at the outer lumen of the selectivity filter. The guanidine group of 1TX adopts a lateral orientation, rather than pointing into the filter as proposed previously. The acidic residues just above the selectivity filter are important in stabilizing the hydrogen-bond network between TTX and Na(v)1.4. The effect of two single mutations of a critical tyrosine residue in the filter of Na(v)1.4 on TTX binding observed experimentally is reproduced using computational mutagenesis. (C)2014 Elsevier Inc. All rights reserved.