The β subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming α subunits, as well as their trafficking and localization. In heterologous expression systems, β1, β2, and β3 subunits influence inactivation and persistent current in different ways. To test how the β4 protein regulates Na channel gating, we transfected β4 into HEK (human embryonic kidney) cells stably expressing NaV1.1. Unlike a free peptide with a sequence from the β4 cytoplasmic domain, the full-length β4 protein did not block open channels. Instead, β4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of β1 or chimeric subunits including the β1 extracellular domain, however, favored inactivation. Coexpressing NaV1.1 and β4 with β1 produced tiny persistent currents, indicating that β1 overcomes the effects of β4 in heterotrimeric channels. In contrast, β1C121W, which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by β4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with β4, persistent current was slightly but significantly increased. Moreover, in β4-expressing neurons fromScn1bandScn1b/Scn2bnull mice, entry into inactivated states was slowed. These data suggest that β1 and β4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted β1 subunits.