Objective  To investigate the effects of rutin (RT) on 5-fluorouracil (5-FU) resistance in human colorectal cancer LoVo/5-FU cells and its possible mechanism.   Methods The drug-resistant LoVo/5-FU cells were established by stepwise exposure to 5-FU. LoVo and LoVo/5-FU cells were cultured in vitro and treated with RT and（or）5-FU for 48 hours, and then the cell viability were detected by cell counting kit-8(CCK-8) assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. The mRNA expression levels of P-glycoprotein (P-gp) and multidrug resistance associated protein 1 (MRP1) were measured by RT-PCR. The protein levels of P-gp, MRP1, phosphorylated protein kinase B(p-Akt), Survivin, cyclin A and cyclin dependent kinase 2 (CDK2) were measured by Western blotting.   Results  LoVo/5-FU cells had a significantly higher protein levels of P-gp, MRP1 and survivin than LoVo cells. The 50% inhibitory concentration(IC₅₀) value of LoVo/5-FU cells to 5-FU and RT was 21.77 mg/L and 98.43 mg/L respectively. Under the influence of RT (10 mg/L), the IC₅₀ value of LoVo/5-FU cells to 5-FU reduced to 10.64 mg/L with a reversal fold of 2.05. RT resulted in an enhance of 5-FU-induced apoptosis and S phase arrest, inactivation of Akt, and lower expression of P-gp, MRP1, survivin, cyclin A and CDK2 in LoVo/5-FU cells.   Conclusion  RT can reverse the drug resistance of LoVo/5-FU cells through down-regulating the expression of drug-resistant related protein and suppressing Akt signaling pathway.