The extracellular matrix (ECM) surrounding cancer cells becomes stiffer during tumor progression, which influences cancer cell behaviors such as invasion and proliferation through modulation of gene expression as well as remodeling of the actin cytoskeleton. In this study, we show that MMP24 encoding matrix metalloproteinase (MMP)-24 is a novel target gene of Yes-associated protein (YAP), a transcription coactivator known as a mechanotransducer. We first examined the effect of substrate stiffness on MMP24 expression in MCF-7 human breast cancer cells and showed that the expression of MMP24 was significantly higher in cells grown on stiff substrates than that on soft substrates. The MMP24 expression was significantly reduced by knockdown of YAP. In contrast, the expression of constitutively active YAP increased MMP24 promoter activity. In addition, binding of YAP to the MMP24 promoter was confirmed by the chromatin immunoprecipitation (ChIP) assay. These results show that ECM stiffening promotes YAP activation, thereby inducing MMP24 expression. Based on the Human Protein Atlas database, breast cancer patients with lower MMP24 expression exhibit the worse survival rates overall. Thus, MMP24 may negatively regulate the aggressiveness of cancer cells under the stiff ECM environment during tumor progression.