Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming alpha-subunit and associated beta-subunits that regulate Nav alpha-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav alpha-subunit also directly affects Navs gating. beta-subunits and glycosylation thus comodulate Nav alpha-subunit gating. We hypothesized that beta-subunits could directly influence alpha-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both beta1- and beta3-subunits coexpression shifted V (1/2) of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 alpha-subunits exist in multiple glycosylated states. The alpha-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when beta1-subunits were coexpressed, suggesting that the beta1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the beta1-subunit increased the expression of this alternative glycosylated form and the beta3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for beta1- and beta3-subunits in the modulation of Nav1.7 alpha-subunit glycosylation and cell surface expression.