Bone disease as a consequence of diabetes mellitus (DM) is not fully understood. The effects of high glucose (30 mM), high insulin (50 nM), or mannitol (30 mM; osmotic control) were evaluated on MC3T3-E1 cells (osteoblasts) in vitro. The mRNA and protein levels of parathyroid hormone (PTH) receptor (PTH1R), collagen I, RANKL, osteoprotegerin (OPG), alkaline phosphatase (ALP), and glucose transporter (GLUT1) were estimated by real-time polymerase chain reaction or Western blotting. The mineralization capacity was analyzed by von Kossa staining. High glucose induced overexpression of RANKL (2×) and OPG (30×), suggesting that RANKL-induced osteoclast activity might not be a dominant mechanism of bone disease in DM, since this increase was followed by increased OPG. Collagen I increased by 12×, indicating an excess of organic matrix production. The expression of ALP decreased by 50%, indicating a deficit in mineralization capacity, confirmed by von Kossa staining. Mannitol induced similar effects as glucose suggesting that extracellular hyperosmolarity was able to stimulate organic matrix production. GLUT1 expression was not altered, and insulin did not reverse most of the effects of glucose, suggesting that glucose uptake by osteoblasts was not altered by high glucose. The data suggest that the bone fragility typical of DM is not a consequence of excessive bone reabsorption but is instead attributable to a defect in organic matrix mineralization. The heightened increase in OPG versus RANKL might cause a decrease in the bone-remodeling cycle. Osteoblasts appear to be more sensitive to extracellular hypertonicity than to the intracellular metabolic effects of hyperglycemia.