Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium- aggravated myotonia, long QT-3 syndrome, and neuropathic pain. This drug elicits tonic block of Na+ channels when cells were stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation- deficient mutant Na+ channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a 1 subunit. The 50% inhibitory concentrations (IC50) for the inactivated block and the resting block of wild-type Na+ channels by mexiletine were measured as 67.8 ± 7.0 µM and 431.2 ± 9.4 µM, respectively (n = 5). In contrast, the IC50 value for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 µM (n = 5), which was within the therapeutic plasma concentration range (2.8-11 µM). Estimated on-rate and off-rate values for the open block of mexiletine at +30 mV were 10.4 µM-1s-1 and 54.4 s-1, respectively. Use-dependent block of mexiletine was greater in inactivation- deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC50 values for the block of persistent late hNav1.4 currents in chloramine-T pretreated cells by mexiletine was 7.5 ± 0.8 µM (n = 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open- channel block of persistent late Na+ currents, which may arise during various pathological conditions.