Background: Diabetic nephropathy (DN), as a complication of diabetes, is featured with hypertension, hyperglycemia, proteinuria and edema. Gypenoside (GP), the main active compound of Gynostemma pentaphyllum, is proved to be effective for DN. In our previous research, we found that GP could protect the glomerulus and reduce proteinuria by up-regulating the expression of nestin and down-regulating TGFB1. However, the panoramic mechanism of GP against DN is still unclear. Objective: This research is designed to reveal the mechanism of GP on DN through network pharmacology and in vivo and in vitro experimental verification. Methods: In this study, active compounds and targets of Gynostemma pentaphyllum were collected from TCMSP. DisGeNET was used for obtaining the targets of DN. The protein-protein interaction network was acquired from the STRING database and analyzed by the MCODE plugin. GO and KEGG enrichment analysis were constructed to explore further information. In vivo and in vitro experiments were also carried out to evaluate the reliability of this study. Western blotting and RT-PCR were used to detect mTOR, 4E-BP1, p70s6k protein expression and Mtor mRNA expression in DN rats, respectively. AKT1, TP53, ESR1 and PTEN protein expression in MPC-5 cells were detected by Western blotting. Results: Twenty-four compounds and 217 targets were selected from Gynostemma pentaphyllum, of which 36 targets overlapped with DN were taken for the potential targets. The results showed that Quercetin, Rhamnazin, Isofucosterol and 3′-methyleriodictyol corresponded to more targets, AKT1, TP53, MYC, ESR1, PTEN were more active. 36 potential targets were mainly involved in autoimmunity, inflammatory response, metabolism and autophagy. In vivo and in vitro experiments showed that GP might protect the podocytes of DN rats by decreasing the protein expression of mTOR, 4EBP1, p70s6k, as well as the mRNA expression of Mtor, and it had the function in regulating the potential targets through decreasing the protein expression of AKT1, TP53 and ESR1 and increasing the expression of PTEN. Conclusion: This research demonstrates that various compounds of Gynostemma pentaphyllum may intervenes in DN through targets of multiple signaling pathways, which involves a large number of biological processes. It can provide novel insights for further research of the mechanism of GP in the treatment of DN.