Dentin matrix phosphoprotein 1 (DMP1) is a non-collagenous, acidic extracellular matrix protein expressed chiefly in bone and dentin. We examined the DMP1 ability to engage cell-surface receptors and subsequently activate intracellular signaling pathways. Our data indeed show that the presence of extracellular DMP1 triggers focal adhesion point formation in human mesenchymal stem cells and osteoblast-like cells. We determine that DMP1 acts via interaction with αvβ3 integrin and stimulates phosphorylation of focal adhesion kinase. Further biochemical characterization confirms the activation of downstream effectors of the MAPK pathways, namely ERK and JNK, after DMP1 treatment. This activation is specifically inhibitable and can also be blocked by the addition of anti-αvβ3 integrin antibody. Furthermore, we show that extracellular treatment with DMP1 stimulates the translocation of phosphorylated JNK to the nucleus and a concomitant up-regulation of transcriptional activation by phosphorylated c-Jun. The evidence presented here indicates that DMP1 is specifically involved in signaling via extracellular matrix-cell surface interaction. Combined with the published DMP1-null data (Feng, J. Q., Ward, L. M., Liu, S., Lu, Y., Xie, Y., Yuan, B., Yu, X., Rauch, F., Davis, S. I., Zhang, S., Rios, H., Drezner, M. K., Quarles, L. D., Bonewald, L. F., and White, K. E. (2006) Nat. Genet. 38, 1310-1315) it can be hypothesized that DMP1 could be a key effector of ECM-osteocyte signaling.