objective: Heterotopic ossification (HO) is a debilitating disease characterized by extraskeletal bone formation. Active TGF-β recruits mesenchymal stromal cells (MSCs), which contribute to trauma-induced HO. Inhibiting TGF-β induced MSC migration and osteogenic differentiation could be a promising treatment for HO. Matrine is an alkaloid from the genus Sophora that can suppress pancreatic and hepatic fibrosis by regulating TGF-β/Smad signaling. We conducted this study to evaluate the effects of matrine on HO and explore the mechanisms, we carried out this study. methods: Achilles tendon puncture was performed in C57BL/6J male mice to establish the HO model. Following treatment with matrine for 3, 6, 9, and 15 weeks, mice were sacrificed and tendons were collected. In vivo, micro-CT, hematoxylin and eosin staining, CD73 and CD90 immunofluorescence, and osteocalcin staining were used to evaluate the development of HO. In vitro, a transwell migration assay was used to evaluate MSC migration. Immunohistochemistry, immunofluorescence and western blotting were used to evaluate the TGF-β/Smad2/3 pathway. Real-time PCR was conducted to analyze the transcription of alkaline phosphatase (Alp), runt-related transcription factor-2 (Runx2), osteocalcin (Ocn), osteopontin (Opn), and type I collagen (Col1). ALP activity and alizarin red staining were used to assess MSC osteogenic differentiation. results: In vivo, matrine significantly reduced ossification and inhibited HO progression. In vitro, matrine significantly suppressed MSC migration, ALP activity, and mineralization of MSCs. Mechanistically, matrine inhibited TGF-β induced Smad2/3 phosphorylation and transcription of Runx2, Alp, and Ocn after osteoinduction. conclusions: Matrine inhibited HO by suppressing the migration and osteogenic differentiation of TGF-β-induced-MSCsin mice.