Mesenchymal stem cells (MSCs) are multipotent cells that have been widely used in cell based transplantation therapy. The use of MSCs requires in vitro expansion in order to fulfill their regenerative capacity. Therefore the proliferative ability of MSCs is one of the key factors which determine MSC therapeutic efficacy. In the present study, we showed for the first time that lithium, a well-known antidepressant, reversibly promoted the proliferation of human bone marrow derived MSCs in vitro. MSCs treated with 5 mm lithium proliferated more rapidly than untreated cells without undergoing apoptosis. Lithium increased the proportion of cells in S phase as well as cyclin D1 expression. Mechanistic studies revealed that these effects were dependent upon the activation of the glycogen synthase kinase 3β (GSK-3β) mediated canonical Wnt pathway. Lithium induced Ser9 phosphorylation, which results in the inhibition of GSK-3β activity, β-catenin accumulation and Wnt pathway activation. Utilizing a specific GSK-3β inhibitor SB216763 or siRNA-mediated inhibition of GSK-3β produced effects similar to those induced by lithium. In contrast, either quercetin, an inhibitor of the β-catenin/TCF pathway, or siRNA-mediated knockdown of β-catenin abolished the proliferative effect of lithium, suggesting that lithium stimulates MSC proliferation via the GSK-3β-dependent β-catenin/Wnt pathway. Collectively, these studies elucidate a novel role of lithium, which may not only provide a simple and effective way to strengthen MSC transplantation therapy efficacy but also shed light on lithium's clinical application for the treatment of certain disorders resulting from β-catenin/Wnt pathway suppression.