Background: Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. Methods: The effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. Results: Real time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content. Conclusions: Our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality. © 2017 The Author(s).