Exposure to high doses of anticancer drugs can induce the emergence of a subpopulation of weakly proliferative and drug-tolerant cells. Drug tolerance can reduce the benefits obtained from canonical treatment and reduce the survival rate of patients. Regulation of SRY-related HMG box transcription factor 4 (SOX4) has been proved to affect drug sensitivity. The current study aimed to explore the role of SOX4 in drug resistance of colorectal cancer (CRC) cells as well as the related molecular mechanisms. Expression patterns of SOX4, microRNA-17 (miR-17), and CYLD in both CRC tissues and cells were determined with their relationship analyzed by bioinformatics analysis, dual-luciferase reporter gene assay, and ChIP. Loss- and gain-function assays were performed to ascertain the effect of SOX4, miR-17, and CYLD on biological cellular processes and drug resistance to 5-FU. SOX4 and miR-17 were found to be highly expressed while CYLD was poorly expressed in CRC tissues and cells. Silencing of SOX4 resulted in the suppression of cellular proliferation, invasion, migration as well as a reduction in CRC drug resistance. Mechanically, CYLD was specifically targeted by miR-17, while SOX4 upregulated the expression of miR-17. Functionally, SOX4 triggered drug resistance of CRC cells to 5-FU through the miR-17/CYLD axis. Taken together, the key findings of the present study provides evidence suggesting that SOX4 elevates miR-17 to decrease CYLD, thus inducing chemotherapy resistance of CRC cells.