Purpose <p>This study aimed to evaluate the potential impact of colorectal cancer stem cell exosomes (CRC CSCs-enriched exosomes/CSCs-EXOs) on drug resistance and cell proliferation of CRC tumor cells.</p> Methods <p>CSCs were enriched from HT-29 cells and characterized by sequential sphere formation, real-time PCR analysis of key stemness genes, and CRC-CSCs markers. The gene expression related to ABC transporters was analyzed in HT-29, HT-29 CSCs, and Caco-2 cells. CSCs-EXOs and parental-EXOs were isolated and characterized from HT-29 cells. The gene expression related to ABC transporters was investigated in Caco-2 cells treated with CSCs-EXOs and parental-EXOs of HT-29 cells by real-time PCR. The survival rate of exosome-treated Caco-2 cells was also studied in the presence of 5-fluorouracil (5-FU) at the IC50 concentration using MTT assay.</p> Results <p>Colonospheres were found to have the ability to form serial spheres, along with the upregulatation of the key stemness genes (<i>p-value</i> ≤ 0.05). The expression of CRC-CSCs markers significantly increased relative to their parental counterparts (<i>p-value</i> ≤ 0.05). Treatment of Caco2 cells with CSCs-EXOs and their parental-EXOs revealed a substantial elevation in expression of drug resistance genes relative to those treated with their parental-EXOs (<i>p-value</i> ≤ 0.0001). The combination treatment of cells with exosomes and 5-FU at the IC50 concentration led to a more pronounced decrease in cell viability in all groups compared to applying 5-FU at the same concentration.</p> Conclusion <p>Our findings underscore the significance of targeting the CSCs-exosome axis as a prospective therapeutic strategy to overcome drug resistance. Upcoming studies ought to concentrate on exploring the molecular machinery of CSCs and tumor cells plasticity through the exosome-mediated functions.</p>