Objective To explore the effects of osmotic pressure on the morphology, biophysical properties, and expression levels of immunophenotype molecules on immature dendritic cells (imDCs) from mechanobiological viewpoint. Methods After treated with different osmotic pressures, the survival rate of DCs was detected using Cell Counting Kit-8 (CCK-8). The changes in morphology and size of DCs were observed under a Laser scanning confocal microscope. Cell electrophoresis was applied to detect the changes in cell electrophoresis mobility, The membrane fluidity of the cells was detected by fluorescence polarization method, and the expression changes of immune-related molecules were detected by real-time fluorescent quantitative PCR (qPCR). Results Both hyperosmosis and hypoosmosis could change the shape and size of cells, even induce apoptosis. The electrophoresis rate of the low permeability group was significantly higher than that of the equal permeability group, while that of the hypoosmosis group was lower (P<0.05). Fluorescence polarization results showed that both hyperosmosis and hypoosmosis could significantly decrease the membrane fluidity of cells (P<0.05). The result of qPCR detection showed that both hyperosmosis and hypoosmosis could significantly increase the expression of CCR7, CD40 ,CD205, CD11a, CD11c on the surface of DCs (P<0.05). Conclusion Hypertonic and hypotonic stress can change the electrophoresis rate, membrane fluidity, and the expression of immune-related molecules, which is important for understanding the immune regulation function of DCs.