Voltage-gated sodium channels (Na v ) consist of a pore-forming α subunit (Na v α) associated with β regulatory subunits (Na v β). Adult skeletal myocytes primarily express Na v 1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na v 1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na v 1.5 currents than those recorded from myoblasts. Unlike myoblasts, I Na recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na v β subunits modulate channel gating and pharmacology, the goal of the present work was to study Na v β subunits during myogenesis. All four Na v β (Na v β1–4) isoforms were present in L6E9 myocytes. While Na v β1–3 subunits were up-regulated by myogenesis, Na v β4 subunits were not. These results show that Na v β genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na + channels during development and myotube formation.