Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD(+) is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD(+) with oscillatory increases of the cytosolic Ca(2+) concentration. The initial Ca(2+) response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD(+) in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD(+) as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.