Triptolide (TP) has an anti-proteinuric effect and is used for the treatment of podocytopathies. TP has also been shown to act directly on immortalized podocytes in culture to protect them from injury. In the present study, we examined the effect of TP on healthy podocytes both in vitro and in vivo to better understand the action of TP on podocytes. We found that treatment of TP at 10 ng/ml, a concentration that is routinely used for podocyte protection, was sufficient to activate pro-apoptotic signaling of MAPK p38, p53 and BAX and induced apoptosis in cultured podocytes; and higher concentrations of TP exacerbated the p38, p53 and BAX activations and apoptosis. Moreover, TP severely downregulated the genes that are essential for podocyte structure and function. Interestingly, in contrast with other agents TP-induced podocyte injury was not prevented by glucocorticoids. In vivo, high-dose TP treatment for prolonged time did not cause podocyte injury, essential genes downregulation, and proteinuria in mice. TP was also not toxic to the podocytes with isolated glomeruli ex vivo. In summary, TP is toxic to immortalized podocytes in culture but not to the podocytes in animals or isolated glomeruli ex vivo. Our study suggests that immortalized podocytes might have genetically evolved to become sensitive to TP toxicity and thus caution should be taken in interpreting data from immortalized podocytes. Nevertheless, in vivo TP could be as safe as glucocorticoids in treating podocytopathies. Finally, TP may be used as a unique in vitro model for studying steroid-resistant podocytopathies.