Chemotherapy is the mainstay for advanced colorectal cancer (CRC) with 5-fluorouracil (5-FU) being the most commonly used chemotherapeutic drug. Many of the patients acquire resistance against 5-FU and hence fail to respond to the treatment. In this study, we investigated the role of exosomes, a class of extracellular vesicles, in the transfer chemotherapeutic drug resistant phenotype between cells. Exosomes were isolated by deferential centrifugation coupled with ultracentrifugation from colorectal cancer cells and were characterized by western blotting, electron microscopy and nanoparticle tracking analysis. Incubation of exosomes secreted by 5-FU resistant CRC cells with parental CRC cells conferred the transfer of drug resistant phenotype. To identify regulators of chemotherapeutic resistance, quantitative proteomics analysis was performed on exosomes secreted by parental and 5-FU resistant CRC cells. Follow up biochemical experiments and qPCR analysis confirmed the role of epithelial-mesenchymal transition (EMT) in this acquired drug resistance. Exosomes isolated from 5-FU resistant CRC cells were intravenously injected into mouse models implanted with parental CRC cells. Follow up treatment with 5-FU confirmed the exosomal transfer of chemoresistance between cells in vivo.