Dystrophin Dp71 is a versatile protein that plays a critical role in regulating diverse processes, including cell adhesion, nuclear architecture, neuronal differentiation, and synapse organization. Moreover, Dp71 is involved in cognitive, retinal vascular and cancer diseases. This multifunctional role is believed to result from the alternative splicing undergoes by Dp71 transcript. As previously outlined, the splicing of exon 78 appears to influence the localization of Dp71 isoforms. Specifically, Dp71d, containing exon 78, shows nuclear localization, while Dp71f, lacking exon 78 and instead having 31 hydrophobic residues (founder sequence), shows cytoplasmic localization. We delineate the mechanism dictating the differential localization between Dp71 isoforms, using various reporter vectors, in combination with protein-protein interaction assays, site-directed mutagenesis on nuclear export signal (NES), knockdown experiments on importin α2 and CRM1 nuclear transporters, and molecular dynamic simulations.