Female-dominant tendinopathies are musculoskeletal disorders caused by repetitive hand posture and motion; they are considered overuse syndromes. Both external mechanical stress and changes in hormone levels might affect disease progression. We have previously reported that estrogen receptor-β (ER)-β expression was associated with the pathogenesis of de Quervain's disease. To study the underlying mechanisms, a cyclic stretching culture system was applied to tendon tissue from ovariectomized (OVX) rats. Furthermore, a collagenase I-induced rat tendinopathy model was established to examine the association of ER-β with disease progression. Our results showed that ER-β expression and the number of apoptotic cells were higher and associated with disease severity in rats with tendinopathy. Mechanical stress altered the morphology of primary tenocytes and collagen fiber alignment in tendons, and up-regulated the expression of matrix metalloproteinase-9, ER-β, and interleukin-1β, as well as induced apoptosis in tenocytes and tendon tissue from OVX rats. This is the first report on the effects of ER-β and mechanical stress in tendinopathy. We hope these findings contribute to new pharmacological therapies targeting ER-β signaling pathways to treat tendon-related diseases.