The regulation of osteoblast proliferation is a key factor in maintaining bone mass. The enhancement of this process can be achieved by stimulating the proliferation of these cells. Mechanical stimulation is one of the important enhancing factors, but the exact cellular mechanisms of mechanical stimulation, i.e., mechanotransduction, are unknown. In order to investigate the role of the cytoskeleton components in mechanotransduction for cell proliferation, I compared the total DNA content in cultured replicates of osteoblast-like cells derived from three human donors following their exposure to enhancing mechanical stimulation, with and without added specific microtubular and microfilament polymerization blockers (Colchicin and Cytochalasin D, respectively). The results revealed the essential and unique role of the microtubular component of the cytoskeleton in mechanotransduction for proliferation by showing that Colchicin blocked the expected increase in the DNA content after mechanical stimulation of the cultured replicates without altering the total DNA content in replicates at static conditions. Conversely, a specific blockage of the microfilament polymerization presented uniform cytotoxic effect in both static and biomechanically active environments. Since previous reports indicated the essential role of microfilament polymerization for the osteoblast metabolic activity, the results of this study further support the hypothesis that the mechanotransduction mechanisms for proliferation and metabolic activity are mediated by different intracellular pathways.