The term tendinopathy indicates a wide spectrum of conditions characterized by alterations in tendon tissue homeostatic response and damage to the extracellular matrix. The current pharmacological approach involves the use of nonsteroidal anti-inflammatory drugs and corticosteroids often with unsatisfactory results, making essential the identification of new treatments. In this study, the pro-regenerative and protective effects of an aqueous fibroin solution (0.5-500 μg/mL) against glucose oxidase (GOx)-induced damage in rat tenocytes were investigated. Then, fibroin anti-hyperalgesic and protective actions were evaluated in two models of tendinopathy induced in rats by collagenase or carrageenan injection, respectively. In vitro, 5-10 μg/mL fibroin per se increased cell viability and reverted the morphological alterations caused by GOx (0.1 U/mL). Fibroin 10 μg/mL evoked proliferative signaling upregulating the expression of decorin, scleraxin, tenomodulin (p < 0.001), FGF-2, and tenascin-C (p < 0.01) genes. Fibroin enhanced the basal FGF-2 and MMP-9 protein concentrations and prevented their GOx-mediated decrease. Furthermore, fibroin positively modulated the production of collagen type I. In vivo, the peri-tendinous injection of fibroin (5 mg) reduced the development of spontaneous pain and hypersensitivity (p < 0.01) induced by the intra-tendinous injection of collagenase; the efficacy was comparable to that of triamcinolone. The pain-relieving action of fibroin (peri-tendinous) was confirmed in the model of tendinopathy induced by carrageenan (intra-tendinous) where this fibrous protein was also able to improve tendon matrix organization, normalizing the orientation of collagen fibers. In conclusion, the use of fibroin in tendinopathies is suggested taking advantage of its excellent mechanical properties, pain-relieving effects, and ability to promote tissue regeneration processes.