A voltage-gated, small, persistent Na+ current ( INa) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent INa that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on INa in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the α-subunit (hH1α) of human cardiac Na+ channels (hNav1.5) plus β1-subunits. Extracellular application of 5 μM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited INa. The late portion of INa ( INa late, measured near the end of each pulse) was almost completely suppressed. INa returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on INa was concentration dependent, with IC50 values of 4.0 ± 0.4 μM for INa peak ( INa peak) and 0.9 ± 0.1 μM for INa late. EPA shifted the steady-state inactivation of INa peak by −19 mV in 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, all at 5 μM concentration, also significantly inhibited INa. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 μM concentration had no effect on INa. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1α induce inactivation-deficient INa and that n-3 PUFAs inhibit mutant INa.