Objective: To investigate the protective effect and mechanism of triptolide on diabetic nephropathy podocytes. Methods: The immortalized glomerular podocytes of mice were cultured in vitro, and stimulating podocytes to induce podocyte injury with high glucose. Triptolide was intervened by real-time quantitative PCR (qRT-PCR) and Western blotting. The effect of triptolide on the expression of Toll-like receptor 4 (TLR4) in podocytes induced by high glucose were detected by qRT-PCR and Western blotting. The overexpressed TLR4 were infected by lentivirus, and the infection effect was detected by qRT-PCR and Western blotting. The proliferation of podocytes in each group was detected by MTT. Flow cytometry was used to detect the apoptosis of podocytes in each group. The levels of IL-6 and TNF-α in the culture supernatant of podocytes of each group were detected by ELISA. Results: qRT-PCR and Western blotting showed that triptolide down-regulated the expression of TLR4 in podocytes induced by high glucose; MTT results showed that triptolide increased the proliferation of podocytes induced by high glucose; the results of cytometry showed that triptolide inhibited the apoptosis of podocytes induced by high glucose; ELISA results showed that triptolide inhibited the secretion of IL-6 and TNF-α by podocytes induced by high glucose; overexpression of TLR4 reversed the protective effect of triptolide on high glucose-induced podocytes. Conclusion: Triptolide can protect the podocytes of diabetic nephropathy by inhibiting TLR4.