N-cadherin, a calcium-dependent cellular adhesive protein, plays important roles during embryonic development and bone formation. The potential of mesenchymal stem cells (MSCs) in osteoblast differentiation and homing to the sites of injury make it a promising cell resource for tissue engineering. However, the role of N-cadherin in MSCs osteoblast differentiation and migration remains still obscure. In the present study, our results showed that prolonged N-cadherin overexpression inhibited osteogenic differentiation of MSCs through negatively regulating β-catenin and ERK1/2 signaling pathways. The mRNA expression levels of osteogenesis-related genes (Osteopontin, Osteocalcin, runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and bone morphogenetic protein 2) were significantly inhibited by N-cadherin, as well as the ALP activity and calcium deposit as stained by Alizarin Red S. While, silencing N-cadherin using shRNA reversed this effect. Furthermore, ectopic bone formation conducted in nude mice verified that N-cadherin significantly inhibited ectopic bone formation of MSCs in vivo. In addition, we also found that the N-cadherin overexpression could promote the migration potential of MSCs. These findings reveal that N-cadherin inhibits osteogenesis but promotes migration of MSCs. The underlying mechanism of N-cadherin inhibiting osteogenesis may through suppressing β-catenin and ERK1/2 signaling pathways.