Rationale: Loss-of-function of the cardiac sodium channel Na^V1.5 causes conduction slowing and arrhythmias. Na^V1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I^Na) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I^Nareduction, but the mechanisms underlying microdomain-specific targeting of Na^V1.5 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 Na^V1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human-induced pluripotent stem cell-derived cardiomyocytes resulted in enhanced whole-cell I^Na, increased action potential upstroke velocity (V^max), and enhanced Na^V1.5 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 Na^V1.5 forward trafficking. Knockout of MAPRE1 in human induced pluripotent stem cell-derived cardiomyocytes led to reduced whole-cell I^Na, decreased V^max, and action potential duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V^maxas well as increased APD. Stochastic optical reconstruction microscopy imaging and macropatch I^Nameasurements 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 Na^V1.5 and I^Napreferentially at the ID region of wild-type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I^Naor Na^V1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of Na^V1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the microtubule plus-end tracking protein EB1 on Na^V1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I^Nawithin the ID region of cardiomyocytes. Graphic Abstract: A graphic abstract is available for this article.