Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.