Objective: Triptolide and celastrol are two most widely studied and promising compounds isolated from Thunder God Vime (Trypterigium wilfordii Hook F) with promising anticancer activity.However, their clinical application was limited by toxicity.We aim to investigate the synergistic anti-proliferative effects of triptolide with celastrol in cancer and the molecular mechanism.Methods: The effects of triptolide and celastrol on cancer cells and the molecular mechanism were assessed by using MTT assay in vitro, xenografts mouse model in vivo, flow cytometry, fluorescence microscope and western blot analysis.Results: The combination of triptolide with celastrol induced strong cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increasing intracellular ROS accumulation in cancer cells.Pretreatment with ROS scavenger N-acetyl-L-cysteine totally blocked the apoptosis induced by triptolide with celastrol.Treatment with celastrol alone decreased many Hsp90 client proteins including Akt,Cdks, EGFR and Raf1, but lead to stress-induced increase of Hsp70 and Hsp27.Adding triptolide to celastrol treatment abrogated this phenomenon and induced more apoptosis.In xenografts mouse model, the lower-dose combination of triptolide with celastrol significant inhibited the growth of tumors without obvious toxicity.Conclusions: Triptolide in combination with celastrol showed outstanding synergistic anticancer effect, suggesting that this beneficial combination may offer a promising treatment option for cancer patients.