Aims: Inactivation of A-type potassium current is a critical determinant of action potential frequency in neurons. In dorsal root ganglion (DRG) neurons, the A-type current is mediated by Kv1.4 channels with "ball and chain" N-type inactivation. Studies have shown that the N-type inactivation of Kv1.4 is subject to modulation by a variety of intracellular factors. We explored how free heme/hemin, which is receiving an increasing attention as a signaling molecule, affects Kv1.4 N-type inactivation. Methods: The effect of hemin on rapidly inactivating native A-type potassium currents in mouse DRG neurons was studied using the whole-cell patch-clamp method and that on rat Kv1.4 (rKv1.4) channels expressed in Xenopus oocytes was examined using the inside-out configuration of the patch-clamp method. To diminish the influence of C-type inactivation, the double mutant K533Y:I535M was utilized. The inside-out patch measurements were carried out with the reducing agent GSH (1 mM) to avoid potential confounding with secondary oxidation caused by hemin. Results: Inclusion of hemin (200 nM) in the whole-cell patch pipette removed fast inactivation of A-type currents in the DRG neurons. In heterologously-expressed rKv1.4 channels, application of 200 nM hemin to the intracellular side progressively removed N-type inactivation following an exponential time course with a time constant of 93±24 s. The apparent EC₅₀ value for hemin-induced removal of inactivation was 18.6±1.0 nM. Extensive wash with hemin-free solution failed to restore inactivation. Protoporphyrin IX and Fe²⁺ had no effect. The effect of hemin on N-type inactivation was abolished by the double mutation C13S:H16A in the ball domain of the channel. Conclusion: Application of hemin selectively disrupts N-type inactivation of Kv1.4 channels by interacting with cysteine and histidine residues in the ball domain. Thus heme is a potent modulator of the Kv1.4 channel and may have a role in regulation of neuronal excitability.