Objective To investigate the effect of C peptide on tubulointerstitial oxidative stress injury in rats with diabetic nephropathy (DN) induced by streptozotocin (STZ). Methods Wistar rats with DN induced by STZ were randomly divided into control group and C peptide group. All rats were treated via intraperitoneal micro-osmotic pump. Normal Wistar rats at the same age were used as the normal group. Blood glucose and24 h urinary albumin were measured before treatment and every 4 weeks during treatment period. After 12 weeks of treatment, the expression of transforming growth factor - β1 (TGF - β1) in renal tubulointerstitium was detected by immunohistochemical staining. Fresh tubulointerstitial tissue homogenate was harvested and the activity of superoxide dismutase (SOD) was detected in WST-1 method, the content of malondialdehyde (MDA) was detected in TBA method, and the expression of protein kinase A (PKA) mRNA was detected by Real-time PCR. Results Levels of blood glucose, 24 h urinary albumin, MDA content and the expression of TGF-β1 were higher in the control group than those in the normal group, while SOD activity decreased and PKA mRNA was downregulated. C peptide treatment did not change blood glucose level but slowed down the increasing of 24 h urinary albumin, reduced the tubulointerstitial TGF-β1 expression and MDA content, increased the SOD activity and upregulated the PKA mRNA expression. Conclusions C-peptide can decrease the tubulointerstitial oxidative stress in DN rats by activating PKA pathway and then improve tubulointerstitial fibrosis and attenuate proteinuria.