Urinary trypsin inhibitor (UTI) is a serine protease inhibitor produced in the liver that inhibits the production and activation of various cytokines, notably transforming growth factor-β (TGF-β), which are associated with the progression of liver fibrosis. However, the various roles of endogenous UTI in liver fibrosis have not been examined. This study, therefore, examined the long-term effects of UTI deficiency during both steady-state conditions and thioacetamide (TA)-induced liver fibrosis. Furthermore, the effects of continuous exogenous UTI administration were examined. Analyses of liver fibrosis marker, hyaluronic acid (HA), TGF-β concentrations and histological findings at 30 weeks of age showed that homozygous UTI-knockout (KO) mice had higher HA and TGF-β concentrations than did heterozygous UTI-KO mice and wild-type mice, although there was no histological evidence of liver fibrosis in these mice. TA treatment for 20 weeks also resulted in greater HA and TGF-β levels in homozygous mice than in heterozygous and wild-type mice. Furthermore, homozygous mice had more severe liver fibrosis based on histological analyses. HA and TGF-β levels were lower in homozygous UTI-KO mice that were continuously administered UTI versus those given distilled water. These findings indicate that UTI deficiency leads to the production of HA and hepatic TGF-β and that administering exogenous UTI can ameliorate these changes. We conclude that endogenous UTI is produced in the liver to suppress the production and activation of TGF-β and that administering exogenous UTI may be therapeutically beneficial for preventing liver fibrosis.