Vascular calcification is a progressive pathology that occurs in many diseases, including atherosclerosis, diabetes, end-stage renal disease and valve disease. This study aimed to investigate whether decorin and transforming growth factor (TGF)-β signal modulation occurs during differentiation of vascular smooth muscle cells (SMCs). Decorin and osteopontin expression was identified in calcified human femoral arteries using immunohistochemistry and biochemical staining. A positive staining pattern of bone-related proteins and mineralisation was also found in aortic root sections from 8–16 week ApoE−/− mice fed a high-fat diet, particularly in the valve leaflets. These data suggest that this model, as well as being an established model of atherosclerosis, is also a suitable model to study vascular calcification. When adenoviral infection of human vascular SMC in vitro was used, decorin overexpression increased SMC osteogenic differentiation fourfold in comparison with controls, as assessed using alizarin red staining and alkaline phosphatase activity. The enhancement of mineralisation was reduced using two approaches to antagonise the TGF-β pathway—namely, adenoviral-mediated overexpression of the latency-associated particle of TGF-β- 1 (LAP-β1) and also a chemical inhibitor of TGF-β type I receptor, SB431542. In conclusion, these results suggest that vascular calcification involves the modulation of the decorin and TGF-β pathway in SMCs. The data may have implications for therapeutic targeting of this devastating pathology.