Voltage-gated cardiac Na⁺ channels are composed of α- and β₁-subunits. In this study β₁-subunit was cotransfected with the α-subunit of the human cardiac Na⁺ channel (hH1_α) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na⁺currents were assessed. Current density was significantly greater in HEK293t cells coexpressing α- and β₁-subunits (I _Na,αβ) than in HEK293t cells expressing α-subunit alone (I _Na,α). Compared withI _Na,α, the voltage-dependent inactivation and activation of I _Na,αβ were significantly shifted in the depolarizing direction. In addition, coexpression with β₁-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n–3)] significantly reduced I _Na,αβ in a concentration-dependent manner and at 5 μM shifted the midpoint voltage of the steady-state inactivation by −22 ± 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n–3)], α-linolenic acid [C18:3(n–3)], and conjugated linoleic acid [C18:2(n–6)] at 5 μM significantly inhibited bothI _Na,αβ and I _Na,α.In contrast, saturated and monounsaturated fatty acids had no effects on I _Na,αβ. This finding differs from the results for I _Na,α, which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of β₁-subunit with hH1_α modifies the kinetics and fatty acid block of the Na⁺ channel.