The activation of orbital fibroblasts can result in fibrosis, finally contributing to thyroid-associated ophthalmopathy (TAO) progression. Although the effect of BTX-A on the treatment of TAO-related strabismus and upper eyelid retraction has long been recognized in clinical work, the underlying mechanism of BTX-A improving TAO-related strabismus and upper eyelid retraction has not been uncovered yet. In the present study, we successfully isolated and authenticated normal and TAO orbital fibroblasts. Compared with PBS, BTX-A and TACA exerted similar inhibitory effects on TAO orbital fibroblast proliferation and ECM production. TGF-beta stimulation induced the proliferation and ECM production by TAO orbital fibroblast, which was significantly inhibited by BTX-A or TACA treatment. Under TGF-beta stimulation, the inhibitory effects of BTX-A or TACA treatment on TAO orbital fibroblast proliferation and ECM production were reversed by TGF-beta/Smad signaling agonist SRI-011381. Collectively, BTX-A inhibited TGF-beta-induced TAO orbital fibroblast activation through inhibiting the TGF-beta/Smad signaling. Considering that TACA shows no satisfactory curative effects on symptoms closely related to the function of extraocular muscles, such as eye movement and diplopia, BTX-A might be a promising agent in TAO treatment.