Triptolide and celastrol are two main active compounds isolated from Thunder God Vine with the potent anticancer activity. However, the anticancer effect of triptolide in combination with celastrol is still unknown. In the present study, we demonstrated that the combination of triptolide with celastrol synergistically induced cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increased intracellular ROS accumulation in cancer cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine dramatically blocked the apoptosis induced by co-treatment with triptolide and celastrol. Treatment with celastrol alone led to the decreased expressions of HSP90 client proteins including survivin, AKT, EGFR, which was enhanced by the addition of triptolide. Additionally, the celastrol-induced expression of HSP70 and HSP27 was abrogated by triptolide. In the nude mice with xenograft tumors, the lower-dose combination of triptolide with celastrol significantly inhibited the growth of tumors without obvious toxicity. Overall, triptolide in combination with celastrol showed outstanding synergistic anticancer effect in vitro and in vivo, suggesting that this beneficial combination may offer a promising treatment option for cancer patients.