INTRODUCTION: CD44 is important in cellular adhesion and invasion, and is upregulated in glioma cells. Here, we extend our previous CD44-antibody blocking and gene silencing experiments to examine, the downstream effects of CD44 knockdown (kd) on the expression of cytoskeletal proteins involved in cellular migration. Atomic force microscope (AFM) was used to measure the stiffness (Young's Modulus, E) of wild type (wt) and CD44-kd glioma cells. METHOD: SNB-19 cells were transfected with either CD44 siRNA, scrambled siRNA, or a non-related gene siRNA. Human non-neoplastic astrocytes were used as a control. Western Blotting (WB), immunocytochemistry (ICC) and flow cytometric analysis were used to analyse changes in cytoskeletal proteins. AFM (CellHesion 200, JPK Instruments) was used to measure E values obtained from force vs. distance plots, using Hertzian mechanics models, by indenting a microsphere (R = 5.6 µm), attached to a AFM cantilever, into cells (10% of cell thickness, nuclear and cytoplasm regions) maintained at 37°C in culture media. RESULTS: Vimentin, GFAP, and microtubule protein expression was associated with lower CD44 protein levels in CD44-kd cells compared to cells with scrambled siRNA. Functionally, CD44-kd cells were less migratory compared to controls. AFM E-values were found to be log-normally distributed, with no differences observed between nuclear and cytoplasm regions. Knockdown CD44 SNB-19 cells (E = 359 ± 180 Pa) were less stiff than wt cells (E = 1099 ± 991 Pa; p = 0.023). The stiffness of wt SNB-19 cells was also greater than control astrocytes (E = 444 ± 311 Pa; p < 0.001). CONCLUSION: The combined results suggest gene silencing of CD44 may be an important therapeutic strategy for reducing adhesion, migration and invasion of glioma cells.