Rationale: Loss-of-function of the cardiac sodium channel NaV1.5 causes conduction slowing and arrhythmias. NaV1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (INa) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific INareduction, but the mechanisms underlying microdomain-specific targeting of NaV1.5 remain largely unknown. Objective: To investigate the role of the microtubule plus-end tracking proteins EB1 (end-binding protein 1) and CLASP2 (cytoplasmic linker associated protein 2) in mediating NaV1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human-induced pluripotent stem cell-derived cardiomyocytes resulted in enhanced whole-cell INa, increased action potential upstroke velocity (Vmax), and enhanced NaV1.5 localization at the plasma membrane as detected by multicolor stochastic optical reconstruction microscopy. Fluorescence recovery after photobleaching experiments in HEK293A cells demonstrated that EB1 overexpression promoted NaV1.5 forward trafficking. Knockout of MAPRE1 in human induced pluripotent stem cell-derived cardiomyocytes led to reduced whole-cell INa, decreased Vmax, and action potential duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and Vmaxas well as increased APD. Stochastic optical reconstruction microscopy imaging and macropatch INameasurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β (glycogen synthase kinase 3β) inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased NaV1.5 and INapreferentially at the ID region of wild-type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell INaor NaV1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the microtubule plus-end tracking protein EB1 on NaV1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of INawithin the ID region of cardiomyocytes. Graphic Abstract: A graphic abstract is available for this article.