OBJECTIVE: This study aimed to explore the main chemical components of Jiangniaosuan Formula (JNSF), the therapeutic effect of JNSF on hyperuricemia (HUA) mice, and the underlying mechanism by which JNSF inhibits renal tubular epithelial cell apoptosis. METHODS: Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (UPLC-Q-TOF-MS) was used to analyze the chemical composition of JNSF and its serum metabolites. Network pharmacology was performed to predict the potential target genes and pathways. In vitro and in vivo models were established to verify the lower serum uric acid (SUA) and renal protective effects. RESULTS: UPLC-Q-TOF-MS identified 61 chemical compounds in JNSF and 56 metabolites in serum after oral administration. Network pharmacology suggested that Hypoxia-Inducible Factor 1-Alpha (HIF-1α), Cysteine-dependent Aspartate-specific Protease-3 (Caspase-3) and B-cell Lymphoma 2 (Bcl-2) might be the therapeutic targets of JNSF for the HUA treatment and JNSF may exert the therapeutic effect on uric acid nephropathy (UAN) through regulating HIF-1α signaling pathway and apoptosis pathway. In vivo experiments showed that JNSF could reduce SUA, protect renal function and tubular function, alleviate renal interstitial edema and fibrosis, reduce the expression of Reactive Oxygen Species (ROS), HIF-1α and Enhancer of Zeste Homolog 2 (EZH2), and inhibit cell apoptosis in HUA mice. In vitro experiments demonstrated that JNSF reversed apoptosis induced by EZH2 overexpression plasmid. Furthermore, we found that UA could promote the binding of HIF-1α to EZH2 protein and its promoter, enhancing EZH2 transcription, suggesting that JNSF could alleviate the progression of HUA-induced kidney injury by inhibiting the activation of ROS/HIF-1α/EZH2 pathway. CONCLUSION: JNSF may attenuate HUA-induced renal injury by inhibiting apoptosis through the downregulation of ROS/HIF-1α/EZH2 pathway.