Objective To establish the rat model of 5/6 nephrectomy-induced chronic renal disease, and investigate the protective effect of Cordyceps sinensis on renal fibrosis. Methods Forty-eight male SD rats were randomly divided into sham-operation group (SO group), 5/6 nephrectomy group (OP group) and5/6 nephrectomy + Cordyceps sinensis treatment group (CS group), with 16 rats in each group. Blood and urine samples were collected 4 weeks and 8 weeks after nephrectomy, and rats were sacrificed for renal tissue samples. Serum creatinine (SCr), blood urea nitrogen (BUN), albumin (Alb) and urinary albumin to creatinine ratio (UACR) were measured, pathological changes and ultrastructure changes of renal tissues were observed, the expression of extracellular matrix fibronectin (FN), collagen type Ⅲ (Col-Ⅲ), fibroblast-specific protein 1 (FSP-1) and α-smooth muscle actin (α-SMA) in renal tissues was detected by immunohistochemistry, and the expression of FN, Col-Ⅲ and α-SMA mRNA in renal tissues was determined by RT-PCR. Results Compared with SO group, significant pathological changes were observed in OP group 4 weeks after surgery, which were characterized by infiltration of inflammatory cells, more deposition of extracellular matrix, podocyte foot process effacement, mitochondrial swelling, vacuolization, distorted cristae and autophagosome formation, and these changes were more significant 8 weeks after surgery. Compared with SO group, SCr, BUN and UACR were significantly higher, Alb was significantly lower, the expression of FSP-1, FN, Col-Ⅲ and α-SMA protein and FN, Col-Ⅲ and α-SMA mRNA in renal tissues was significantly higher in OP group 4 weeks after surgery, and these differences were more significant 8 weeks after surgery. Compared with OP group, the pathological changes of renal tissues obviously lessened, mitochondrial damage significantly alleviated, UACR significantly decreased, Alb significantly increased, and the expression of FSP-1, FN, Col-Ⅲ and α-SMA protein and FN, Col-Ⅲ and α-SMA mRNA in renal tissues significantly decreased in CS group 8 weeks after surgery. Conclusion Cordyceps sinensis ameliorates 5/6 nephrectomy-induced renal fibrosis of rats by relieving mitochondrial damage and reducing deposition of ECM.