OBJECTIVES: Celastrol has shown therapeutic effects in diabetic nephropathy (DN). This study aimed to elucidate its underlying mechanisms through bioinformatics analysis and experimental validation. METHODS: Differentially expressed genes (DEGs) between DN and control groups were obtained from GSE30122 and GSE30528 datasets. Target genes of Celastrol were collected from relevant biological databases and intersected with the DEGs. Functional enrichment analysis was conducted to explore the associated biological processes. Immune cell infiltration in DN was analyzed, and a Lasso regression model was constructed to identify DN-associated gene markers with diagnostic potential. The binding affinity of celastrol to target proteins was evaluated using molecular docking. Additionally, high glucose (HG)-treated human kidney 2 (HK-2) cells were subjected to cell viability assays, flow cytometry, ELISA, and immunoblotting. RESULTS: A total of 69 key target genes of celastrol were identified, primarily involved in oxidative stress, inflammation, and Phosphoinositide 3-Kinase (PI3K)/Protein Kinase B (Akt) signaling pathways. Immune cell infiltration analysis revealed significant differences in CD4 CONCLUSION: Celastrol exerts protective effects on DN by modulating key molecular pathways, particularly those involved in inflammation and oxidative stress.