Abstract: OBJECTIVE To investigate the role of celastrol in TNF-α-induced cell death in colon cancer. METHODS SW480 cells were treated with TNF-α combined with celastrol. After that, MTT assay was performed to measure the cell death; Annexin Ⅴ/PI staining was performed to detect the cell apoptosis; Western blot analysis was performed to evaluate the cleavage of caspase-8, caspase-9, caspase-3, and the expression of CYLD; Co-immunoprecipitation was performed to detect the deubiquitination of RIP1 protein. RESULTS Combination with celastrol significantly promoted the cell death and apoptosis induced by TNF-α treatment in SW480. Combination with celastrol significantly promoted the cleavage of caspase-8, caspase-9, and caspase-3 induced by TNF-α treatment. Meanwhile, the activation of caspase-8 is earlier than the activation of caspase-9 and caspase-3. The results of co-immunoprecipitation and western blot indicated that the deubiquitination of RIP1 in TNF-α-treated SW480 cells were significantly enhanced due to the combination of celastrol. In addition, the results of western blot assay demonstrated that the expression of CYLD could be significantly up-regulated due to the celastrol treatment. Moreover, the transfection of CYLD siRNA abolished the synergistic effect on TNF-α-induced cell death in SW480 cells. CONCLUSION Celastrol enhances the TNF-α-induced apoptosis by promoting the deubiquitination of RIP1 in colon cancer.