Introduction <p>Hyperglycemia increases the risk of bone fragility by promoting reactive oxygen species and advanced glycation end products, which disrupt osteoblast activity. Mechanical stress, including osmotic stress from elevated glucose levels, affects bone homeostasis; however, the specific impact of osmotic stress on osteoblast function is not fully understood. The transient receptor potential vanilloid 4 (TRPV4) channel, known to mediate calcium influx in response to mechanical stress, plays a key role in osteoblast differentiation. This study investigated the effects of osmotic stress on osteoblast differentiation and mineralization, as well as the role of TRPV4-mediated calcium influx.</p> Materials and methods <p>We investigated the effects of osmotic stress on osteoblast differentiation and mineralization using MC3T3-E1 cells. Mannitol and sorbitol treatments were adjusted to match the osmolality of glucose to assess osmotic stress effects. TRPV4 involvement was examined using the TRPV4 antagonist, HC-067047.</p> Results <p>Mineralization was inhibited not only by glucose, but also by treatment with mannitol and sorbitol, which were adjusted to match the osmolality of glucose. Both glucose and mannitol treatments inhibited the nuclear translocation of Runx2 while decreasing the mRNA expression of osteogenic markers such as osteocalcin, ALP, and collagen I. Notably, osmotic stress suppressed calcium influx through TRPV4 channels, which is linked to differentiation induction. Furthermore, HC-067047 inhibited mineralization and osteogenic marker expression.</p> Conclusion <p>These findings indicate that osmotic stress impairs osteoblast mineralization by inhibiting TRPV4-mediated calcium influx. TRPV4 may represent a therapeutic target for mitigating bone loss in diabetes, suggesting a novel approach for treating diabetic bone disease.</p>