Critical illness myopathy is a disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that a shift in the voltage dependence of fast inactivation of sodium currents contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid-denervated; SD). In the current study we examined whether expression of Nav1.5 contributes to the altered voltage dependence of sodium channel inactivation in SD muscle. We used TTX and μ-conotoxin GIIIB to selectively block Nav1.4 in SD muscle and found that the level of Nav1.5 did not correlate closely with the shift in fast inactivation. Surprisingly, we found that the voltage dependence of inactivation of Nav1.4 was similar to that of Nav1.5 in skeletal muscle in vivo. In severely affected fibres, inactivation of both Nav1.4 and Nav1.5 was shifted towards hyperpolarized potentials. We examined the role of denervation and steroid treatment in the shift of the voltage dependence of inactivation and found that both denervation and steroid treatment contribute to the shift in inactivation. Our results suggest that modulation of the voltage dependence of inactivation of both Nav1.4 and Nav1.5 in vivo contributes to loss of electrical excitability in SD muscle.