The transformation of tunica albuginea-derived fibroblasts (TAFs) into myofibroblasts plays an important role in the pathological progress of Peyronie's disease (PD). However, no treatment which addresses this transformation is currently available. Estrogen has been shown to inhibit the progression of fibrosis in a number of fibrotic diseases. The aim of this study was to determine whether estrogen [17β‑estradiol (E2)] suppresses the diffentiation of primary rat TAFs into myofibroblasts in vitro. TAFs obtained from male Sprague‑Dawley rats were stimulated with either transforming growth factor‑β1 (TGF‑β1) or E2. Western blot analysis and immunofluorescence staining were used to assess changes in the expression levels of α‑smooth muscle actin (αSMA). The expression levels of additional proteins (GAPDH, p‑Smad2, Smad2, Smad4, RhoA, Rac1, ROCK1 and ROCK2) were also measured by western blot analysis. We used collagen gel assays to assess cell contractility. Additionally, the concentration of hydroxyproline in the TAF cell culture medium was detected using commercially available kits. We found that E2 reduced αSMA expression which was induced by TGF‑β1. E2 also suppressed the TGF‑β1‑induced increase in the concentration of hydroxyproline (a marker of collagen) in addition to suppressing the contraction of TAFs. The key processes affected by TGF‑β1 treatment included the phosphorylation of Smad2, ras homolog gene family, member A (RhoA) and Rho‑associated, coiled-coil containing protein kinase 2 (ROCK2); this increase in phosphorylation was inhibited by treatment with E2. Collectively, these results demonstrate that by modulating the activation of the TGF‑β1‑Smad and RhoA‑ROCK2 signaling pathways, E2 inhibited the transformation of TAFs into myofibroblasts, decreased the expression of collagen and suppressed the contraction of myofibroblasts in response to TGF-β1 stimulation.