Transdifferentiation of vascular smooth muscle cells (VSMC) into chondrogenic cells contributes significantly to vascular calcification during the pathogenesis of atherosclerosis. However, the transcriptional mechanisms that control such phenotypic switch remain unclear. This process is characterized by the induction of Sox9 and Col2a1 genes accompanied by the repression of myocardin (Myocd) and SMC differentiation markers such as SM22, SM α-actin and SM-MHC. Here we explore the regulatory role of SOX9, the master regulator for chondrogenesis, in modulating SMC marker gene expression. qRT-PCR and luciferase assays show that over-expression of SOX9 inhibits SMC gene transcription and promoter activities induced by myocardin, the master regulator of smooth muscle differentiation. Such suppression is independent of the CArG box in the SMC promoters but dependent on myocardin. EMSA assay further shows that SOX9 neither participates in SRF (serum response factor) binding to the CArG box nor interacts with SRF, while co-immunoprecipitation demonstrates an association of SOX9 with myocardin. Conversely, myocardin suppresses SOX9-mediated chondrogenic gene Col2a1 expression. These findings provide the first mechanistic insights into the important regulatory role of SOX9 and myocardin in controlling the transcription program during SMC transdifferentiation into chondrocytes.