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Diabetes nephropathy (DN) is one of the most common causes of end stage renal disease (ESRD) globally. Medication options to stop or slow the progression of chronic renal disease (CKD) are limited, and patients with DN remain at a high risk of developing renal failure. Inonotus obliquus extracts (IOEs) of Chaga mushroom have been shown to have anti-glycemic, anti-hyperlipidemia, antioxidant, and anti-inflammatory effects against diabetes. In this study, we examined the potential renal protective role of an ethyl acetate layer after water-ethyl acetate separation from Inonotus obliquus ethanol crude extract (EtCE-EA) from Chaga mushrooms in diabetic nephropathy mice after preparation with 1/3 NT + STZ. Our data showed that treatment with EtCE-EA can effectively regulate blood glucose, albumin-creatinine ratio, serum creatinine, and blood urea nitrogen (BUN) levels, and it can improve the renal damage in 1/3 NT + STZ-induced CRF mice with an increase in concentration (100, 300, and 500 mg/kg). In the immunohistochemical staining test, EtCE-EA can effectively reduce the expression of TGF-β and α-SMA after induction according to the increase in the concentration (100 mg/kg, 300 mg/kg), thereby slowing down the degree of kidney damage. Our findings demonstrate that EtCE-EA could provide renal protection in diabetes nephropathy, possibly due to the decreased expression of transforming growth factor-β1 and α-smooth muscle actin. [ABSTRACT FROM AUTHOR]