Mechanical vibrations are potent regulators of bone formation in vivo and in vitro, promoting osteoblast differentiation. Despite extensive research on the cellular responses to vibration, the underlying mechanisms remain unclear. In this study, the effects of mechanical vibrations on osteoblast differentiation and nuclear displacement were examined, focusing on the dynamics of the cell nucleus, actin stress fibers, and focal adhesions, which connect the nucleus to the extracellular matrix. Alkaline phosphatase gene expression, an osteoblast differentiation marker, was significantly upregulated in MC3T3-E1 cells at 50 Hz compared to 12.5 and 100 Hz (p < 0.05) under 0.5 G. The frequency-dependent response was independent of vibration direction, as confirmed using an exciter in horizontal or vertical vibration, and also independent of fluid shear stress in medium, as validated by observing medium sloshing using slow-motion imaging. Nuclear displacement under horizontal vibration (0.5 G) was analyzed across 20–70 Hz at 10-Hz intervals, and peaked at 40–50 Hz with significant increases at 40 Hz vs. 20/30 Hz and 50 Hz vs. 20 Hz (p < 0.05). These findings indicate a distinct correlation between osteoblast differentiation and nuclear displacement, implying that mechanical vibrations modulate cellular differentiation by altering actin stress fiber and focal adhesion dynamics in a frequency-dependent manner. This is supported by an elastic model estimating actin stress fiber tension based on observed nuclear displacement. This study offers new insights into the frequency dependence of osteoblast differentiation and its mechanotransduction mechanism, and supports the development of optimized mechanical stimulation therapies for bone regeneration.