The regulation of the extracellular matrix (ECM) by mechanical stress is of interest as the ECM is essential in the development of pelvic organ prolapse. In the present study, the effect of overexposure to mechanical stress on the ECM, and the probable underlying mechanisms in cultured human uterosacral ligament fibroblasts (hUSLFs), was explored. Mechanical stress has an effect on oxidation‑antioxidation products in parametrial ligament fibroblasts. Thus, hUSLFs were incubated with different concentrations of hydrogen peroxide to elucidate any potential interactions. Excess mechanical stress and H2O2 inhibited cell proliferation, and decreased mRNA and protein expression levels of ECM components, collagen 1, collagen 3 and elastin. Further analysis revealed that the mRNA expression level of matrix metalloproteinase‑2 (MMP‑2) was increased and TIMP metallopeptidase inhibitor 2 (TIMP‑2) decreased, and in addition the MMP2/TIMP2 mRNA ratio was increased, which may facilitate the degradation of the ECM. Due to the key role of the transforming growth factor β1 (TGF‑β1)/mothers against decapentaplegic homolog 2 (Smad2) signaling pathway in fibrosis, the present study investigated the effect of excess mechanical stress and H2O2 on TGF‑β1/Smad2 signaling. The results indicated that excess mechanical stress and H2O2 treatment suppressed phosphorylated Smad2 expression and decreased the levels of TGF‑β1. Activation of the TGF‑β1 signaling pathway by either mechanical stress or H2O2 was demonstrated to attenuate cell proliferation and ECM components, and also increased the MMP2/TIMP2 mRNA ratio. These findings suggested that mechanical stress and H2O2 overexposure inhibit cell proliferation and remodel the ECM network via regulation of the TGF‑β1 signaling pathway.