The aim of the present study was to examine the protective effects of the urinary trypsin inhibitor ulinastatin (UTI) on renal interstitial inflammation and fibrosis in rats subjected to unilateral ureteral obstruction (UUO). A total of 24 male Wistar rats were randomly divided into the three groups; the sham operation (SOR) group (n=8), the UUO group (n=8) and the UUO+UTI group (post‑UUO UTI treatment, n=8). UUO was performed with complete ligation of the left ureter. As a medical intervention, saline (4 ml kg‑1 d‑1) and UTI (40000 units kg‑1 d‑1) were injected, respectively, into the animals of the corresponding groups on day one following surgery. The rats in all three groups were euthanized on day seven post surgery. Blood samples were harvested for blood urea nitrogen (BUN) and serum creatinine (Scr) content measurements. The degree of interstitial pathological changes in the tissues from the obstructed kidneys were observed through hematoxylin and eosin (H&E) and Masson staining. The CD68+ macrophage amount, tumor necrosis factor‑α (TNF‑α), interleukin 1β (IL‑1β), nuclear factor‑κB (NF‑κB), transforming growth factor‑β1 (TGF‑β1) and type I collagen (Col‑I) levels were examined immunohistochemically. The protein expression levels of NF‑κB were examined using western blot analysis. Total superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of homogenates were measured spectrophotometrically. The results revealed that ulinastatin had no statistically significant effect on the BUN and Scr levels (P>0.05). However, in comparison with the SOR group, the UUO group exhibited significantly more severe renal interstitial pathological injury in terms of tubular dilation, epithelial atrophy, renal interstitial inflammatory cell infiltration and proliferation of fibrous tissues, as well as significantly elevated levels of interstitial CD68+ macrophages, IL‑1β, TNF‑α, NF‑κB, TGF‑β1 and Col‑I (P<0.01). UTI treatment significantly reduced UUO‑induced renal interstitial damage with reduced levels of interstitial CD68+ macrophages, IL‑1β, TNF‑α, NF‑κB, TGF‑β1 and Col‑I and MDA (P<0.05), and increased SOD levels (P<0.05). In conclusion, the present study indicated that UTI is able to effectively inhibit UUO‑side renal interstitial inflammatory reaction and fibrosis in UUO‑inflicted rats.