Objective To observe the influence of renal sympathetic denervation (RSD) on renal interstitial fibrosis and transforming growth factor beta 1(TGF-β1) and microRNA-21 (miR-21) in rats with unilateral ureteral obstruction(UUO). Methods 40 male Wistar rats were randomly divided into UUO group (A group, n=10), sham UUO group (B group, n=10), RSD+UUO group (C group, n=10) and RSD+sham UUO group (D group, n=10). Rats in A group and C group underwent unilateral ureteral ligation, while those in B group and D group underwent sham operation. Rats in C group and D group were followed by RSD. Rats were sacrificed at 21 days after the operation to evaluate the fibrosis by Masson staining. Immunohistochemical staining and Western blotting were used to detect the expressions of collagen I (COL-I), collagen Ⅲ(COL-Ⅲ) and TGF-β1 in four groups. The expression of miR-21 was detected by fluorescence in situ hybridization (FISH) and quantitative real-time PCR (RT-qPCR). Results A large amount of collagen deposition was observed in the renal interstitial area in A and C group compared to either B or D group (P＜0.05), but the change in C group was decreased significantly than that in A group (P＜0.05). Similarly, the expressions of COL-I, COL-Ⅲ, TGF-β1 and miR-21 were obviously higher in A and C group compared to either B or D group (P＜0.05), but those change in C group were decreased significantly than those in A group (P＜0.05). The above indexes were not significantly different between B group and D group (P＞0.05). Conclusion RSD may relieve the renal interstitial fibrosis in UUO rats, and down-regulate the expression of TGF-β1 and miR-21.