Voltage-gated sodium channels (Nav) consist of a pore-forming α subunit (Navα) associated with β regulatory subunits (Navβ). Adult skeletal myocytes primarily express Nav1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Nav1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Nav1.5 currents than those recorded from myoblasts. Unlike myoblasts, INa recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Navβ subunits modulate channel gating and pharmacology, the goal of the present work was to study Navβ subunits during myogenesis. All four Navβ (Navβ1–4) isoforms were present in L6E9 myocytes. While Navβ1–3 subunits were up-regulated by myogenesis, Navβ4 subunits were not. These results show that Navβ genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na+ channels during development and myotube formation.