Objective: To investigation the action and mechanism of macrophage migration inhibition factor (MIF) in drug resistance of colorectal cancer. Methods: The 5-fluorouracil (5-FU)-resistant human colon cancer LoVo/5-FU cells were established by stepwise exposure of human colon cancer LoVo cells to increasing concentrations of 5-FU. The studied parameters included the sensitivity of cells to 5-FU (IC50), MIF protein expression, glucose uptake ability of cells, lactic dehydrogenase (LDH) activity of cells and lactate production from cultured cells supernatant, which were detected by CCK-8 assay, Western blot analysis, 2-NBDG method, microporous assay and kit assay, respectively. The differences in above parameters between LoVo/5-FU cells and their parent LoVo cells were compared, and changes in these parameters in LoVo/5-FU cells before and after MIF interference and overexpression by siRNA and lentivirus transfection, or inhibition of aerobic glycolysis by PFKFB3 inhibitor PFK-15 were examined. Results: The LoVo/5-FU cells were successfully constructed, which showed significantly increased MIF protein expression, IC50 to 5-FU, glucose uptake, LDH activity and lactate production level compared with their parent LoVo cells (all P<0.05). In LoVo/5-FU cells, the IC50 to 5-FU, glucose uptake, LDH activity and lactate production level were significantly decreased after MIF interference by siRNA, while those above parameters were significantly increased after MIF overexpression (all P<0.05). After inhibition of aerobic glycolysis by PFK-15, the IC50 to 5-FU, glucose uptake and lactate production level in LoVo/5-FU cells were significantly decreased (all P<0.05), but the LDH activity showed no significant change (P>0.05), and the same effects were exerted by PFK-15 in LoVo/5-FU cells with MIF overexpression (all P<0.05). Conclusion: MIF enhanced the resistance of LoVo cells to 5-FU by increasing the aerobic glycolysis.