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We have recently shown that ABCG2-rich extracellular vesicles (EVs) form between neighbor breast cancer cells and actively concentrate various chemotherapeutics, resulting in multidrug resistance (MDR). Here we studied the signaling pathway regulating ABCG2 targeting to EVs as its inhibition would overcome MDR. The PI3K-Akt signaling pathway was possibly implicated in subcellular localization of ABCG2; we accordingly show here that pharmacological inhibition of Akt signaling results in gradual re-localization of ABCG2 from the EVs membrane to the cytoplasm. Cytoskeletal markers including β-actin and the tight junction protein ZO-1, along with the EVs markers ABCG2 and Ezrin–Radixin–Moesin revealed that this intracellular ABCG2 retention leads to gradual decrease in the size and number of EVs, resulting in EVs elimination and complete reversal of MDR. Inhibition of Akt signaling restored drug sensitivity to mitoxantrone and topotecan, bona fide ABCG2 transport substrates, hence being equivalent to MDR reversal achieved with the ABCG2 transport inhibitor Ko143. Remarkably, apart from loss of ABCG2 transport activity, treatment of MCF-7/MR cells with Ko143 resulted in cytoplasmic re-localization of ABCG2, similarly to the phenotype observed after Akt inhibition. We conclude that the PI3K-Akt signaling pathway is a key regulator of subcellular localization of ABCG2, EVs biogenesis and functional MDR. Furthermore, proper folding of ABCG2 and its targeting to the EVs membrane are crucial components of the biogenesis of EVs and their MDR function. We propose that Akt signaling inhibitors which disrupt ABCG2 targeting and EVs biogenesis may readily overcome MDR thus warranting in vivo studies with these promising drug combinations.