Laminin-5 (laminin-332) is an important basement membrane protein that regulates cell attachment and motility. Recent studies have shown that laminin-5 is expressed in human mesenchymal stem cells (MSCs) in culture and that the laminin gamma2 chain is transiently expressed in chondrocytes during development. These studies suggest that laminin-5 may be involved in the regulation of chondrogenic differentiation of MSCs. In this study, we examined a possible role of laminin-5 in the proliferation and differentiation of human MSCs. When MSCs were incubated in the presence of a coated or soluble form of laminin-5 in a growth medium, they proliferated more rapidly than nontreated cells, keeping their differentiation potential. On the other hand, laminin-5 potently suppressed the chondrogenic differentiation of MSCs. These activities were mediated mainly by integrin alpha3beta1. However, laminin-5 had no effect on the osteogenic differentiation of MSCs. These results suggest that laminin-5 may contribute to the development of bone tissues by promoting the proliferation and by suppressing the chondrogenic differentiation of MSCs.