Background: Cisplatin is an important agent in first-line chemotherapy against gastric cancer (GC). However, consequential drug resistance limits its effectiveness for the treatment of GC. Exosomes which are loaded with proteins, lipids and RNAs, have been proven to transfer malignant phenotype. This study aims to explore the role and mechanism of exosomal RPS3 protein in transmitting a chemoresistance phenotype from cisplatin resistant to cisplatin sensitive gastric cancer cells.Methods: A cisplatin resistant gastric cancer cell line SGC7901R was established by continuously grafting SGC7901S cells into cisplatin-containing culture medium. Exosomes from SGC7901R and SGC7901S were obtained and confirmed through ultracentrifuge and Nano Analyzer. By LC-MS/MS analysis methods, we detected the differentially expressed proteins in SGC7901R cells exosomes and SGC7901S cells exosomes. Western blotting was used to verify the differential expression of exosomal RPS3 between cisplatin resistant and parental cell lines. Subsequently, a series of in vitro assays and a xenograft tumor model were used to observe the functions of exosomal RPS3 protein in GC.Results: SGC7901R cell derived exosomes were readily taken up by cisplatin sensitive SGC7901S cells, thus triggering off a phenotype of chemoresistance in the receptor cells. Subsequently, it was demonstrated that exosomal RPS3 was essential for inducing chemoresistance of receptor cells as shown by the acquisition of this phenotype in SGC7901S cells with enforced expression of RPS3. Further mechanism study demonstrated that cisplatin‑resistant gastric cancer cells-derived exosomal RPS3 enhanced the chemoresistance of cisplatin‑sensitive gastric cancer cells through the PI3K-Akt-cofilin-1 signaling pathway.Conclusion: Cisplatin resistant gastric cancer cells communicate with sensitive cells through the intercellular delivery of exosomal RPS3 and activation of the PI3K-Akt-cofilin-1 signaling pathway. Targeting exosomal RPS3 protein in cisplatin resistant gastric cancer cells may thus be a promising strategy to overcome cisplatin resistance in gastric cancer.