Voltage-gated sodium channel Na1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Na1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Na1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Na1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Na1.6-specific blockers. Structural mapping of disease-related Na1.6 mutations provides insights into their pathogenic mechanism.