Objective: To investigate the effect of Berberine on renal tubulointerstitial injury and its potential mechanism in rats with type 2 diabetes mellitus (T2DM). Methods: Thirty Sprague-Dawley rats were randomly divided into 3 groups: normal control rats (NC group), diabetic rats without drug treatment (DM group), diabetic rats treated with Berberine (BBR group) for 8 weeks. At the end of the study, blood and urine samples were collected for biochemical examination, and tubulointerstitial fibrosis was quantified by Hematoxylin and Eosin (HE) and Masson staining. The expressions of E-cadherin (E-cad), α-smooth muscle actin (α-SMA), nuclear factor-κB (NF-κB) and monocyte chemoattractant protein 1 (MCP-1) were detected by immunohistochemistry analysis, real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Results: 24 h urinary microalbumin (mAlb)[(170.5±58.1) vs (253.7±53.0) mg]and urinary N-acetyl-glucosaminidase (NAG)[(33.5±7.2) vs (49.5±9.3)U/L]in diabetic rats were significantly decreased by BBR treatment(both P<0.05). The apparent renal tubulointerstitial injury was found in the DM group, which was ameliorated by BBR treatment. The expression of α-SMA, NF-κB and MCP-1 were significantly decreased, accompanied by increased expression of E-cad in BBR-treated DM rats (all P<0.05). Conclusion: BBR could ameliorate renal tubulointerstitial injury in diabetic rats, the mechanism of which may be associated with the amelioration of epithelial-mesenchymal transition (EMT) through suppressing the expression of the NF-κB and MCP-1.