Transforming growth factor (TGF)-β1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-β1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-β1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-β1 plays a role in the development of equine endometrosis. In Exp. 1, endometrial expression of α-SMA in different stages of endometrosis was determined. In endometrial tissues from the mid luteal phase (n = 6 for each stages of endometrosis) and the follicular phase of the estrous cycle (n = 5 for each stages of endometrosis), mRNA transcription and protein expression of α-Sma were evaluated by Real-time PCR and Western-blot, respectively. The α-Sma mRNA transcription and protein expression levels were correlated with the severity of endometrosis (P < 0.05). In both phases of the estrous cycle, α-SMA protein expression was up-regulated in final stage of endometrosis compared to initial stage (P < 0.05). In Exp. 2, the dose- and time-dependent effects of TGF-β1 on expression of α-SMA and ECM components were determined, as well as cell proliferation of equine fibroblasts. Equine endometrial fibroblasts (n = 6, Kenney and Doig category I) were stimulated with vehicle or TGF-β1 (1, 5, 10 ng/ml) for 24, 48 or 72 h. Then, mRNA transcription of α-Sma, collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin 1 (Fn1) were determined by Real-time PCR. The production of ECM components was determined by ELISA. Transforming growth factor-β1 increased the mRNA transcription of α-Sma and ECM components in a dose- and time-dependent manner in cultured endometrial fibroblasts (P < 0.05). Additionally, TGF-β1 at a dose of 10 ng/ml increased α-SMA protein expression and COL1, COL3, FN production after 72 h of stimulation (P < 0.05). The data showed a positive linkage between the presence of myofibroblasts and severity of endometrosis. We conclude that TGF-β1 may participate in pathological fibrotic changes in equine endometrial tissue by induction of myofibroblast differentiation, increased production of ECM components and fibroblast proliferation.