Cartilage Link Protein 1 (Crtl1) is an extracellular matrix (ECM) protein that stabilizes the interaction between hyaluronan and versican and is expressed in endocardial and endocardially-derived cells in the developing heart, including cells in the atrioventricular (AV) and outflow tract (OFT) cushions. Previous investigations into the transcriptional regulation of the Crtl1 gene have shown that Sox9 regulates Crtl1 expression in both cartilage and the AV valves. The cardiac transcription factor Mef2c is involved in the regulation of gene expression in cardiac and skeletal muscle cell lineages. In this study we have investigated the potential role of Mef2c in the regulation of ECM production in the endocardial and mesenchymal cell lineages of the developing heart. We demonstrate that the Crtl1 5' flanking region contains two highly conserved Mef2 binding sites and that Mef2c is able to bind to these sites in vivo during cardiovascular development. Additionally, we show that Crtl1 transcription is dependent on Mef2c expression in fetal mitral valve interstitial cells (VICs). Combined, these findings highlight a new role for Mef2c in cardiac development and the regulation of cardiac extracellular matrix protein expression.