Saffron is not only a precious spice used in the international market, but also a traditional Chinese herbal medicine. The petals of saffron (SP) have been reported to possess multiple pharmacological activities such as antioxidant, anti-inflammatory, and anti-hyperuricemia (HUA) activities. Our previous studies showed that the flavonoid extract of SP had ameliorative effects on HUA animals. However, its underlying pharmacological mechanism remains unclear. The aim of this study is to elucidate the molecular targets and potential mechanism of SP in treating HUA by combining network pharmacology and experimental validation. A network pharmacology approach was used to conduct target prediction, PPI network analysis, GO enrichment, and KEGG analysis to predict the potential targets and signaling pathways of SP against HUA. Furthermore, the potential mechanism of SP against HUA was verified using a molecular docking method and in vivo experiments. Based on the network pharmacological analysis, a total of 32 common targets between SP and HUA were screened, among them, ABCG2, URAT1, GLUT9, PYGL, and PTGS2 were identified as the key targets. KEGG enrichment analysis further showed that the PI3K-Akt and NF-KB signaling pathways might be involved in the mechanisms of SP against HUA. Molecular docking results showed that ABCG2, URAT1, and GLUT9 were well connected to five potential pharmacodynamic components of SP. In vivo experiment results revealed that the SP flavonoid extract reduced UA level, improved renal function and enhanced antioxidant activities of HUA rats. Western blot and real-time quantitative polymerase chain reaction (RT-qPCR) methods confirmed that the SP flavonoid extract regulated the expressions of ABCG2, URAT1 and GLUT9. By combining network pharmacology with experimental verification, SP may effectively treat HUA by regulating urate transporters ABCG2, URAT1, and GLUT9 related to the PI3K/Akt/NF-KB signaling pathway. These findings provide an experimental foundation for the development and clinical utilization of SP. (c) 2025 Published by Elsevier B.V. on behalf of SAAB.