5-Fluorouracil (5-FU) is the preferred chemotherapeutic drug used in the treatment of colon cancer; however, drug resistance affects its clinical efficacy. Visfatin, an adipokine that promotes tumour development, has the potential to increase resistance to chemotherapy. The present study aimed to verify the effects of visfatin on the sensitivity of colon cancer cells to 5-FU and to elucidate the potential mechanisms involved. Tissue microarrays (TMAs) were used to analyse visfatin differential expression in normal colon and colon cancer tissues, and the data were further validated in vitro. Cell Counting Kit-8, clone formation, caspase-3/7 activity assays, as well as other analyses were used to verify the effects of visfatin on sensitivity to 5-FU. TMA and correlation analyses were used to predict and verify the correlation between visfatin and stromal cell-derived factor-1 (SDF-1). Rescue experiments and PI3K/Akt inhibitors were used to verify the role of the visfatin/SDF-1/Akt axis in the sensitivity of colon cancer cells to 5-FU. Visfatin was found to be highly expressed in colon cancer tissues and cell lines. Moreover, visfatin knockdown increased apoptosis, reduced cell proliferation and enhanced the chemosensitivity of DLD-1 and SW48 cells to 5-FU. A positive correlation between visfatin and SDF-1 was observed, with the knockdown of visfatin enhancing cell sensitivity to 5-FU chemotherapy by targeting the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) axis. Furthermore, the Akt signalling pathway downstream of SDF-1/CXCR4 proved to be critical in the decreased sensitisation of colon cancer cells to 5-FU induced by visfatin. On the whole, the present study demonstrates that visatin can potentially decrease colon cancer cell apoptosis, promote proliferation and decrease colon cancer cell sensitivity to 5-FU via the visfatin/SDF-1/Akt axis.