Objective From the perspective of Biophysics and immunology, the effects of Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA)on the biophysical characteristics, cytoskeleton and migration ability of mouse derived dendritic cells (DCs) were analyzed, so as to explore the effect of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) on the immune function of DCs and its potential mechanism. Methods The bone marrow-derived monocytes from C57BL/6J mice were isolated and induced to differentiate into immature dendritic cells (imDCs) by 20 ng/mL recombinant mouse granulocyte colony stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4). After 6 days, 100 ng/mL lipopolysaccharide was added to induce mature dendritic cells (mDCs). Further the morphological observation and positive rate of CD11c in imDCs and mDCs were analyzed, Then in different concentrations of EPA and DHA (0~60 μM), the cell viability and apoptosis of DCs were detected by cell conting kit-8 (CCK-8) kit and flow cytometry. After the optimal concentration of EPA and DHA were determined, the changes of the membrane fluidity, electrophoretic mobility (EPM) and osmotic fragility of DCs were separately detected by the fluorescence polarization, cell electrophoresis and concentration gradient. The expression of filamentous actin (F-actin) was detected by the immunofluorescence. Finally, the migration ability of DCs was detected by the Transwell system. Results The positive rate of CD11c in DCs was about 80%, the viability of DCs were decreased in a dose-dependent manner under the action of EPA and DHA of different concentrations (P<0.05 or P<0.01), which didn’t induce the apoptosis of DCs. Under the action of EPA and DHA(50 μM), the osmotic fragility and EPM of DCs were significantly decreased (P<0.05 or P<0.01), and the membrane fluidity were significantly increased (P<0.05 or P<0.01). Meanwhile, the expression of F-actin in DCs were obviously increased (P<0.05 or P<0.01), and the migration rate of cells were obviously decreased (P<0.05 or P<0.01). Conclusion ω-3 PUFAs may affect the cytoskeleton structure and biophysical characteristics of DCs, inhibit the migration, and then affect its immune function. It not only provides the theoretical backup to deeply understand the mechanism of ω-3 PUFAs on immune function of DCs, but also possibilities for the application of immunosuppressants in the autoimmune diseases and organs transplantation.