Objective: To investigate the effects of acupotomy on skeletal muscle fibrosis and collagen deposition in a rabbit knee osteoarthritis (KOA) model. Methods: Rabbits (n = 18) were randomly divided into control, KOA, and KOA + acupotomy (Apo) groups (n = 6). The rabbits in the KOA and Apo groups were modeled using the modified Videman's method for 6 weeks. After modeling, the Apo group was subjected to acupotomy once a week for 3 weeks on the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, and anserine bursa tendons around the knee. The behavior of all animals was recorded, rectus femoris tissue was obtained, and histomorphological changes were observed using Masson staining and transmission electron microscopy. The expression of transforming growth factor-β1 (TGF-β1), Smad 3, Smad 7, fibrillar collagen types I (Col-I) and III (Col-III) was detected using Western blot and real-time polymerase chain reaction (RT-PCR). Results: Histological analysis revealed that acupotomy improved the microstructure and reduced the collagen volume fraction of rectus femoris, compared with the KOA group (P = .034). Acupotomy inhibited abnormal collagen deposition by modulating the expression of fibrosis-related proteins and mRNA, thus preventing skeletal muscle fibrosis. Western blot and RT-PCR analysis revealed that in the Apo group, Col-I, and Col-III protein levels were significantly lower than those in the KOA group (both P < .01), same as Col-I and Col-III mRNA levels (P = .0031; P = .0046). Compared with the KOA group, the protein levels of TGF-β1 and Smad 3 were significantly reduced (both P < .01), as were the mRNA levels of TGF-β1 and Smad 3 (P = .0007; P = .0011). Conversely, the levels of protein and mRNA of Smad 7 were significantly higher than that in the KOA group (P < .01; P = .0271). Conclusion Acupotomy could alleviate skeletal muscle fibrosis and delay KOA progress by inhibiting collagen deposition through the TGF-β/Smad pathway in the skeletal muscle of KOA rabbits.