Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death worldwide. About 61% of patients are diagnosed at an advanced stage of CRC. Chemotherapy is currently the mainstay for advanced CRC with 5-FU being the most commonly used chemotherapeutic drug. While some patients respond to chemotherapy treatment others don’t referred to as inherent resistance. Among the responding patients some acquire resistance and therefore fail to further respond to treatment. In this study, we investigated whether exosomes, a class of extracellular vesicles, can transfer chemotherapeutic drug resistance between cells. Exosomes were isolated from colorectal cancer cells resistant to 5-FU and characterised by Western blotting and electron microscopy. Furthermore, exosomes secreted by resistant cells were incubated with SW620 and HCT116 CRC cells. FACS apoptosis assay confirmed that the CRC cells incubated with exosomes were protected with 5-FU induced cell death. To identify regulators of chemotherapeutic resistance, label-free quantitative proteomic analysis was performed on exosomes isolated from parental and resistant cells. The analysis revealed the differential abundance of many proteins implicated in epithelial-mesenchymal transition (EMT) including YBX1. Overall, this study highlights the role of exosomes in the transfer of chemotherapeutic drug resistance.