A voltage-gated small persistent Na⁺ current has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent Na⁺ current that may cause arrhythmias. In the present study, we investigated the effects of the n-3 polyunsaturated fatty acids (PUFAs) on the Na⁺ current (I_Na) in HEK293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁ subunits. Extracellular application of eicosapentaenoic acid (EPA, C20:5n-3) at 5 µM significantly inhibited I_Na. The late portion (I_Na,late, measured near the end of each pulses) of I_Na was almost completely suppressed. I_Na returned to the pre-treated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration-dependent with the IC₅₀ values of 4.0 ± 0.4 µM for the peak (I_Na,peak) of I_Na and 0.9 ±0.1 µM for the I_Na.late. EPA shifted the steady-state inactivation of I_Na,peak by -19 mV to the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, at 5 µ also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids, stearic acid and palmitic acid, at 5 µ had no effects on I_Na. Our data demonstrate that the double mutations at 409 and 410 sites in D1-S6 region of hH1 induce inactivation-deficient Na⁺ currents and the n-3 PUFAs inhibit the mutant Na⁺ current.