BACKGROUND: Induction of cholestasis is one of the methods of liver fibrosis which causes the development of oxidative stress, increased expression of fibrogenic markers, excessive deposition of extracellular matrix, and finally the incidence of fibrosis. Plantago ovata is known as a rich source of various secondary metabolites such as phenolic compounds, flavonoids, alkaloids, trypanoids, and ascorbic acid. OBJECTIVES: the present study, the expression of TGF- β as a fibrotic marker and serum alkaline phosphatase (ALP) changes in cholestatic rats treated with P. ovata extract were evaluated. METHODS: In this study, 48 adult Wistar rats were used. The rats were randomly divided into eight groups of six animals each as follows: (1) healthy control group without bile duct ligation (BDL) surgery and treatment; (2–4) three healthy experimental plus P. ovata groups: rats without BDL, treated with P. ovata at dose levels of 100, 200, and 400 mg/kg body weight, respectively; (5) the BDL group: rats with BDL and treated with distilled water; and (6–8) the BDL plus P. ovata groups: rats with BDL and treated with P. ovata at dose levels of 100, 200, and 400 mg/kg body weight, respectively. The rats were treated with P. ovata extract for 45 consecutive days (once per day). After euthanasia and serum isolation, ALP enzyme level was measured. Moreover, the rat liver was fixed in 10% formalin buffer solution. The immunohistochemical study was performed by TGF-β antibody. Data analysis was performed using the One-way ANOVA and Kruskal-Wallis test and the Prism statistical program (p <0.0001). RESULTS: The results showed a significant increase in the serum levels of ALP enzyme and TGF-β expression in BDL group. Treatment with P. ovata extract was able to significantly improve these changes in a dose-dependent manner. CONCLUSIONS: The results of this study showed that P. ovata extract probably due to its phenolic compounds and its antioxidant effect has a protective effect on the liver and subsequently improves the increased serum ALP level and also reduced TGF-β expression