The long-term effects of palmitate (PA) on osteogenic differentiation capacity of human mesenchymal stromal cells (hMSCs) were investigated by cultivating the cells in osteogenic differentiation medium (O-w/o) and osteogenic medium containing PA (O-BSA-PA) for 21 days. Osteogenic medium containing BSA (O-BSA) was used as a control. By means of rt-qPCR, successful osteogenic differentiation was observed in the O-w/o regarding the levels of osteogenic and cell-communication related genes (OCN, Col1, BMP2, ITGA1, ITGB1, Cx43, sp1) in contrast to expression levels observed in cells incubated within basal medium. However, in the O-BSA, these genes were found to have decreased significantly. In cases of Cx43 and sp1, PA significantly reinforced the reductive effect of BSA alone. O-BSA notably decreased glucose and pyruvate consumption, whereas glutamine consumption significantly increased. In comparison to O-BSA addition of PA significantly reduced glycolysis and glutaminolysis. ToF-SIMS analysis confirmed increased incorporation of supplemented deuterated PA into the cell membranes, while the overall PA-concentration remained unchanged compared to cells incubated in the O-BSA and O-w/o. Therefore, the effects on gene expression and the metabolism did not result from the membrane alterations, but may have risen from inter- and intracellular effects brought on by BSA and PA.