Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. Their abnormal functions are associated with numerous diseases, such as epilepsy, cardiac arrhythmia, and pain syndromes. Therefore, these channels represent important drug targets. Even in the post-resolution revolution era, a lack of structural information continues to impede structure-based drug discovery. The limiting factor for the structural determination of Nav channels using single particle cryo-electron microscopy (cryo-EM) resides in the generation of sufficient high-quality recombinant proteins. After extensive trials, we have been successful in determining a series of high-resolution structures of Nav channels, including NavPaS from American cockroach, Nav1.4 from electric eel, and human Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7, with distinct strategies. These structures established the framework for understanding the electromechanical coupling and disease mechanism of Nav channels, and for facilitating drug discovery. Here, we exemplify these methods with two specific cases, human Nav1.4 and Nav1.7, which may shed light on the structural determination of other membrane proteins.