Introduction: Cardiovascular diseases (CVDs) are a major global health concern, often involving the dysfunction and remodelling of blood vessels. The sonic hedgehog (SHh) signalling pathway has been implicated in the regulation of vascular cell fate during development and disease. This study aimed to investigate the role of SHh signalling in the myogenic differentiation of mesenchymal stem cells (MSCs) and adventitial progenitor cells (APCs) into vascular smooth muscle cells (SMCs) in vitro. Methods: Mouse bone marrow-derived MSCs and rat Sca1+ adventitial progenitor cells were cultured and treated with recombinant SHh (r-SHh) protein, as well as SHh pathway inhibitors cyclopamine, HPI-4, and anti-Patched1 antibody. Cell viability was assessed using the Alamar Blue assay. The expression of SMC differentiation markers calponin (CNN1) and myosin heavy chain 11 (MYH11) was evaluated by immunocytochemistry, Western blotting, and real-time qRT-PCR. Results: Treatment with r-SHh significantly increased the expression of CNN1 and MYH11 in both MSCs and APCs, as demonstrated by immunocytochemistry and Western blot analysis. This effect was attenuated by the SHh pathway inhibitors cyclopamine, HPI-4, and anti-Patched1 antibody. Real-time qRT-PCR further confirmed the up-regulation of the SHh target gene Gli1 and the SMC marker CNN1 in response to r-SHh, which was reduced by cyclopamine treatment. Discussion and Conclusion: The results of this study indicate that SHh signalling plays a crucial role in promoting the myogenic differentiation of MSCs and APCs into vascular SMCs in vitro. Targeting the SHh pathway may offer a potential therapeutic strategy for the management of cardiovascular diseases characterised by aberrant vascular remodelling.