Background and Purpose: The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav1.1–Nav1.7. Experimental Approach: Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings. Key Results: The inhibitory effects of TTX on Nav1.1–Nav1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav1.7 T1425M and Nav1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively. Conclusions and Implications: The reduced binding of chiriquitoxin to Nav1.7 was attributed to its C11–OH and/or C12–NH2, based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.