Objective ·To investigate the protective effect of the natural compound hypericin (HYP) on podocyte injury in diabetic nephropathy (DN) and its underlying molecular mechanism. Methods ·A total of eighteen db/db mice were selected to establish the DN model and were randomly divided into three groups: the model group, the db/db + HYP low-dose group (db/db+HYP-L, 1.5 mg/kg), and the db/db+HYP high-dose group (db/db+HYP-H, 3 mg/kg). Additionally, six db/m mice were designated as the normal control group. The following parameters were measured in mice from each group: fasting blood glucose (FBG), insulin (INS), total cholesterol (TC), triglyceride (TAG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), serum creatinine (Scr), blood urea nitrogen (BUN), urinary albumin-to-creatinine ratio (UACR), as well as levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Renal pathological changes and podocyte ultrastructure in mice from each group were examined by hematoxylin-eosin (HE) staining, periodic acid-Schiff (PAS) staining, and transmission electron microscopy. The expression of the podocyte marker Wilms tumor 1 (WT-1) in renal tissues from each group was assessed using immunohistochemistry and Western blotting. The protein expression level of nuclear factor-κB (NF-κB) was also measured by Western blotting. Human podocyte lines were cultured in vitro to establish a high-glucose-induced podocyte injury model. The experiment was divided into five groups: normal glucose (5.5 mmol/L glucose, NG), mannitol (5.5 mmol/L glucose+24.5 mmol/L mannitol, MA), high glucose (30.0 mmol/L glucose, HG), high glucose+0.05 μg/mL HYP, and high glucose+0.20 μg/mL HYP. Cells were harvested after 48 h of incubation. The protein levels of WT-1, phospho-NF-κB p65 (p-NF-κB p65), and NF-κB p65 were determined by Western blotting. The mRNA expression levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) in human podocytes across all groups were measured by quantitative real-time PCR (qPCR). Results ·Compared with the model group, HYP administration significantly reduced the levels of FBG, INS, TC, TAG, LDL, Scr, BUN, and UACR in db/db mice, while significantly increasing the level of HDL (all P<0.05). Concomitantly, HYP alleviated podocyte injury in db/db mice, as evidenced by a marked reduction in foot process effacement and restoration of a comb-like alignment of the foot processes. Both in vivo and in vitro studies demonstrated that HYP upregulated the expression of WT-1 while concurrently suppressing the phosphorylation of NF-κB p65 and the subsequent release of NF-κB-mediated inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) (all P<0.05). Conclusion ·HYP alleviates podocyte injury in db/db mice, an effect that may be attributed to the upregulation of WT-1 expression and inhibition of the NF-κB signaling pathway, leading to reduced expression of downstream inflammatory cytokines.