Diabetic nephropathy (DN) is one of the main complications of diabetes. It is closely associated with the dysfunction of glomerular endothelial cells (GECs) under hyperglycemia. Severe inflammation is an important inducer for the development of GECs dysfunction, and it contributes to the disruption of tight junctions in GECs and the increased endothelial permeability. Sinomenine, an alkaloid monomer extracted from the rhizome of Sinomenium acutum, is recognized for its multiple pharmacological functions, including an anti-DN property. The present study aimed to explore the potential functional mechanism of Sinomenine against DN. Animals were randomly divided into Sham, DN, DN + Sinomenine (20 mg/kg), and DN + Sinomenine (40 mg/kg) groups. The Sinomenine or vehicle was administered every day for 6 weeks, followed by collecting renal tissues for further detection. Increased body weights, elevated blood glucose levels and UAE values, aggravated renal tissue pathology, higher concentrations of IL-18 and IL-1 beta in renal tissues, and reduced claudin-5 expression were observed in DN rats. However, the administration of Sinomenine significantly alleviated all these DN-related changes. Furthermore, human renal glomerular endothelial cells (HrGECs) were treated with high glucose (HG, 30 mM) with or without Sinomenine (50, 100 mu M) for 24 h. We found that Sinomenine treatment ameliorated the elevated production of IL-18 and IL-1 beta, increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) value, and reduction of claudin-5 and C/EBP-alpha in HG-treated HrGECs. Moreover, the regulatory effect of Sinomenine on endothelial monolayer permeability in HG-treated HrGECs was abolished by the knockdown of C/EBP-alpha, indicating C/EBP-alpha is required for the effect of Sinomenine. We concluded that Sinomenine alleviated diabetic nephropathy-induced renal glomerular endothelial dysfunction via activating the C/EBP-alpha/claudin-5 axis.