The voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. Being associated with a variety of channelopathies, they are targeted by multiple pharmaceutical drugs and natural toxins. We determined the crystal structure of a bacterial Nav channel NavRh in a potentially inactivated state a few years ago, which is a homotetramer in primary sequence but exhibits structural asymmetry. Employing the modern methods of cryo-EM, we recently determined the near atomic resolution structures of a Nav channel from American cockroach (designated NavPaS) and from electric eel (designated EeNav1.4). These structures reveal the folding principle and structural details of the single-chain eukaryotic Nav channels that are distinct from homotetrameric voltage-gated ion channels. Unexpectedly, the two structures were captured in drastically different states. Whereas the structure of NavPaS has a closed pore and four VSDs in distinct conformations, that of EeNav1.4 is open at the intracelluar gate with VSDs exhibiting similar “up”states. The most striking conformational differenc occurs to the III-IV linker, which is essential for fast inactivation. The III-IV undergoes a pronounced repositioning from NavPaS to EeNav1.4, resulting in the insertion of the IFM fast inactivation motif on the III-IV linker into the corner enclosed by the S4-S5 and S6 segments in repeats III and IV of EeNav1.4. Based on the structural features, we suggest an allosteric blocking mechanism for fast inactivation of Nav channels by the IFM motif. Structural comparison of the conformationally distinct EeNav1.4 and NavPaS provides important insights into the electromechanical coupling mechanism of Nav channels.