Myxomatous mitral valve disease, the most common form of canine heart disease, causes mitral regurgitation due to valvular degeneration (proliferation and thickening of spongiosa), similar to mitral valve prolapse in humans. The bioactive fragments cleaved from the fibrous components are called matricryptins. Arresten (α1 chain) and canstatin (α2 chain), the cleaved fragments of type IV collagen, are known as basement membrane-derived matricryptins which are expressed in the cardiac tissue in rats. Although the expression of these matricryptins may be altered during mitral valve degeneration, it has not been clarified. In this study, we investigated the expression of the matricryptins during mitral valve thickening in rats and its role in valvular interstitial cells (VICs). The expression of canstatin was increased in the thickened mitral valve in the old rats (>36-weeks-old). Stimulation of cultured rat mitral valve with transforming growth factor (TGF)-β increased the thickness of mitral valve and the expression of arresten. Both canstatin and arresten affected TGF-β-induced activation of VICs (increase of α-smooth muscle actin expression). These results suggest that the expression of canstatin and arresten changes with thickening of the rat mitral valve and that they are involved in the activation of VICs.