Objective: To investigate the reversal effect of neferine (Nef) on oxaliplatin (OXA)-resistance in human colon cancer cells and the mechanism. Methods: Human colon cancer HCT116 cells were cultured in a step-wised exposure to increasing concentrations of OXA (2, 4, 8, 12, 24 and 48 μmol/L) to induce and create the OXA-resistant cell line HCT116/OXA; the cytotoxic effect of Nef on HCT116/OXA cells was evaluated, and its optimal treatment concentration and time were determined; in HCT116/OXA cells after exposure to OXA (IC50 concentration) alone, Nef (optimal treatment concentration) alone or OXA (IC50 concentration) plus Nef (optimal treatment concentration), the proliferation and apoptosis as well as the expressions of apoptosis-associated proteins (Bcl-2, Bax, PARP and p-PARP) were analyzed and compared. Results: The resistance of HCT116/OXA cells to OXA was significantly increased compared with the parent HCT116 cells (IC50: 21.00 μmol/L vs. 112.00 μmol/L, P<0.05), with a resistance index of 5.33. Nef showed significant inhibitory effect on proliferation of HCT116/OXA cells in a concentration-dependent manner (P<0.05), and its optimal treatment concentration and time was 5 μmol/L and 24 h, respectively; compared with lone OXA treatment, the tolerance of HCT116/OXA cells to OXA plus Nef treatment was significantly reduced (IC50: 112.00 μmol/L vs. 45.47 μmol/L, P<0.05), and the reverse index was 2.46. Nef alone exerted no significant effect on apoptosis of HCT116/OXA cells (P>0.05), but the apoptosis inducing effect on HCT116/OXA cells by its combination with OXA was significantly greater than that by OXA alone treatment (P<0.05); compared with Nef or OXA alone treatment, the expression level of anti-apoptotic protein Bcl-2 was significantly decreased, and the expression levels of apoptotic protein Bax and p-PARP were significantly increased in HCT116/OXA cells after OXA plus Nef treatment (all P<0.05). Conclusion: Nef can reverse OXA-resistance in HCT116/OXA cells, and the mechanism may be associated with its regulating Bcl-2/Bax expression, and thereby, producing a synergistic effect with OXA.