Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. Populations of the garter snake, Thamnophis sirtalis, have evolved TTX resistance by substituting amino acid residues in the highly conversed domain IV P-loop of NaV1.4, allowing them to feed on tetrodotoxic newts. Different populations of the garter snake have different degrees of TTX-resistance closely related to the number of amino acid substitutions. Here, we tested the voltage dependence, kinetics, and ion selectivity of NaV1.4 containing sequences from different garter snake populations. Using Xenopus oocytes under cut-open voltage clamp, we observed significant changes in voltage dependent gating properties of the TTX resistant NaV. The most TTX-resistant channel had hyperpolarized activation midpoint (−6 mV) compared to TTX-sensitive channels. Fast inactivation of the TTX-resistant NaV had significant depolarizing shifts of 2.2 mV in mildly TTX-resistant channels, and 4.5 mV in strongly TTX-resistant channels. Depolarizing shifts in slow inactivation were also observed in TTX-resistant channels with 20 mV and 16 mV shifts in mildly resistant and strongly resistant channels, respectively. In addition, ion selectivity and permeability of TTX-resistant channels were significantly different from those of the TTX-sensitive channel. These results suggest TTX resistance comes at a cost to channel performance caused by changes in the gating properties and ion selectivity of TTX-resistant sodium channels.Supported by a NSERC Discovery Grant to PCR.