In response to sustained depolarization or prolonged bursts of activity in spiking cells, sodium channels enter long-lived non-conducting states from which recovery at hyperpolarized potentials occurs over hundreds of milliseconds to seconds. The molecular basis for this slow inactivation remains unknown, although many functional domains of the channel have been implicated. Expression studies in Xenopus oocytes and mammalian cell lines have suggested a role for the accessory beta1 subunit in slow inactivation, but the effects have been variable. We examined the effects of the beta1 subunit on slow inactivation of skeletal muscle (NaV1.4) sodium channels expressed in HEK cells. Co-expression of the beta1 subunit impeded slow inactivation elicited by a 30-s depolarization, such that the voltage dependence was right shifted (depolarized) and recovery was hastened. Mutational studies showed this effect was dependent upon the extracellular Ig-like domain, but was independent of the intracellular C-terminal tail. Furthermore, the beta1 effect on slow inactivation was shown to be independent of the negative coupling between fast and slow inactivation.