RLIP76 is an anti-apoptotic transporter, participating in the multi-specific drug transport and resistance. In the absence of chemotherapy drugs, the knockout or inhibition of RLIP76 leads to pronounced tumor regression. RLIP76 transports anthracycline and vinca alkaloid drugs and mediates the resistance to these drugs. However, functions of RLIP76 in drug resistance colorectal cancer remain unclear. HCT-8 and the vincristine (VCR)-resistant colorectal cancer cell line HCT-8/VCR (HCT-8/V) were used in the present study. The effects of RLIP76 knockdown by the lentivirus were examined in cultured cells, including growth, apoptosis, invasion, and signaling pathways by qRT-PCR, western blot analysis and transwell assay. The relative level of RLIP76 in HCT-8 and HCT-8/V was assessed by western blot analysis, finding RLIP76 was overexpressed in HCT-8/V. Then, HCT-8/V cancer cells were transfected with lentivirus encoding RLIP76-specific shRNA (KD) and the control (NC), and no significant difference of RLIP76 level between the NC cells and cells without transfection was found, but the relative mRNA level decreased to 0.277±0.016 and protein level also reduced in KD cells. Cell functions changed after RLIP76 knockdown in HCT-8/V. The IC50 of VCR decreased from 164.4±1.734 to 13.95±2.008 (µg/ml) (p<0.05) in cell culture. The cell number reduced from 329.67±20.23 to 176.33±2.52 (p<0.05) in migration assay and from 294.67±30.07 to 153±22.11 (p<0.05) in invasion assay. Moreover, apoptotic proteins, including cleaved-caspase-8, cleaved-caspase-9, cleaved-Parp and Bax increased. The phosphorylation level of Erk also reduced significantly. The present study showed that RLIP76 is a key effector of cancer cell survival, invasion, and migration and possibly an important target to improve drug resistance and tumor treatment.