Background: The bone synthesizing function of osteoblasts (OBs) is a highly demanding energy process that requires nutrients. However, how nutrient availability affects OBs behavior and bone mineralization remain to be fully understood. Methods: MC3T3-E1 cell line and primary OBs (OBs) cultures were treated with physiological levels of glucose (G; 5.5 mM) alone or with the addition of palmitic acid (G+PA) at different concentrations. Mitochondria morphology and activity were evaluated by fluorescence microscopy, qPCR, and oxygen consumption rate (OCR) measurement, and OBs function was assessed by mineralization assay. Results: The addition of non-lipotoxic levels of 25 μM PA to G increased mineralization in OBs. G+25 μM PA exposure reduced mitochondria size in OBs, which was associated with increased activation of dynamin-related protein 1, a mitochondrial fission protein, enhanced mitochondria OCR and ATP production, and increased expression of oxidative phosphorylation genes. Treatment with Mdivi-1, a putative inhibitor of mitochondrial fission, reduced osteogenesis and mitochondrial respiration in OBs. Conclusions: Our results revealed that OBs function was enhanced in the presence of glucose and PA at 25 μM. This was associated with increased OBs mitochondrial respiration and dynamics. These results suggest a role for nutrient availability in bone physiology and pathophysiology.