PURPOSE: This research aims to investigate the role and potential mechanisms of Aloin in Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) through network pharmacology and experimental approaches. METHODS: Using network pharmacology methods, potential targets of Aloin and targets related to CP/CPPS were screened from public databases. The protein-protein interaction (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to predict the core targets and pathways of Aloin against CP/CPPS. The effects of Aloin in ameliorating CP/CPPS were verified in animal experiments. RESULTS: A total of 235 genes interacting with Aloin in CP/CPPS were identified. PPI network analysis revealed five core targets: AKT1, EGFR, ESR1, HSP90AA1, and SRC. GO analysis yielded 2916 enrichment results, with 2562 related to Biological Process (BP), 94 to Cellular Component (CC), and 260 to Molecular Function (MF). KEGG pathway analysis identified 172 pathways. Molecular docking confirmed stable binding between Aloin and core targets. Molecular dynamics simulations further validated binding stability by analyzing Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (Rg), hydrogen bonds, Solvent Accessible Surface Area (SASA), and Gibbs free energy of Aloin-target complexes. Experimental validation showed that Aloin alleviated pain, reduced inflammatory factors, and decreased oxidative stress in a rat model of CP/CPPS. The qRT-PCR results showed that Aloin intervention reduced the mRNA expression of AKT1, EGFR, HSP90AA1, and SRC, while increasing ESR1 mRNA expression. These changes may underlie its therapeutic effects in CP/CPPS. CONCLUSION: Our study revealed that Aloin exerts a beneficial effect on mitigating the pain symptoms associated with CP/CPPS, ameliorating inflammation, and reducing oxidative stress. Through network pharmacology, potential targets and signaling pathways were identified, suggesting the therapeutic promise of Aloin for CP/CPPS. These findings advocate for further exploration into its clinical efficacy and mechanistic underpinnings in the treatment of CP/CPPS.