BACKGROUND Jianpi Gushen Huayu Decoction (JPGS) has been used to clinically treat diabetic nephropathy (DN) for many years. However, the protective mechanism of JPGS in treating DN remains unclear. AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN. METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model. We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics. Furthermore, we examined the effects of JPGS on c-Jun N-terminal kinase (JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-kappa B)/NOD-like receptor family pyrin domain containing 3 (NLRP3). RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress. Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice. A total of 51 differential metabolites were screened. Pathway analysis results indicated that nine pathways significantly changed between the control and model groups, while six pathways significantly altered between the model and JPGS groups. Pathways related to cysteine and methionine metabolism; alanine, tryptophan metabolism; aspartate and glutamate metabolism; and riboflavin metabolism were identified as the key pathways through which JPGS affects DN. Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-kappa B/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors. CONCLUSION JPGS could markedly treat mice with streptozotocin (STZ)-induced DN, which is possibly related to the regulation of several metabolic pathways found in kidneys. Furthermore, JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-kappa B/NLRP3 pathway and inhibit JNK/P38 pathway-mediated apoptosis in DN mice.