Rationale: Pyridine nucleotides regulate the cardiac Na + current ( I Na ) through generation of reactive oxygen species (ROS). Objective: We investigated the source of ROS induced by elevated NADH. Methods and Results: In human embryonic kidney (HEK) cells stably expressing the cardiac Na + channel, the decrease of I Na (52±9%; P <0.01) induced by cytosolic NADH application (100 μmol/L) was reversed by mitoTEMPO, rotenone, malonate, DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), PK11195, and 4′-chlorodiazepam, a specific scavenger of mitochondrial superoxide and inhibitors of the mitochondrial complex I, complex II, voltage-dependent anion channels, and benzodiazepine receptor, respectively. Anti–mycin A (20 μmol/L), a complex III inhibitor known to generate ROS, decreased I Na (51±4%, P <0.01). This effect was blocked by NAD + , forskolin, or rotenone. Inhibitors of complex IV, nitric oxide synthase, the NAD(P)H oxidases, xanthine oxidases, the mitochondrial permeability transition pore, and the mitochondrial ATP-sensitive K + channel did not change the NADH effect on I Na . Analogous results were observed in cardiomyocytes. Rotenone, mitoTEMPO, and 4′-chlorodiazepam also blocked the mutant A280V GPD1-L (glycerol-3-phosphate dehydrogenase 1-like) effect on reducing I Na , indicating a role for mitochondria in the Brugada syndrome caused by this mutation. Fluorescent microscopy confirmed mitochondrial ROS generation with elevated NADH and ROS inhibition by NAD + . Conclusions: Altering the oxidized to reduced NAD(H) balance can activate mitochondrial ROS production, leading to reduced I Na . This signaling cascade may help explain the link between altered metabolism, conduction block, and arrhythmic risk.