Sonoporation mediated by microbubbles has being extensively studied as a promising technique to facilitate gene/drug delivery to cells. Previous studies mainly explored the membrane-level responses to sonoporation. To provide in-depth understanding on this process, various sonoporation-induced cellular responses (e.g., membrane permeabilization and cytoskeleton disassembly) generated at different impact parameters (e.g., acoustic driving pressure and microbubble-cell distances) were systemically investigated in the present work. HeLa cells, whose α-tubulin cytoskeleton was labeled by incorporation of a green fluorescence protein (GFP)-α-tubulin fusion protein, were exposed to a single ultrasound pulse (1 MHz, 20 cycles) in the presence of microbubbles. Intracellular transport via sonoporation was assessed in real time using propidium iodide and the disassembly of α-tubulin cytoskeleton was observed by fluorescence microscope. Meanwhile, the dynamics of an interacting bubble-cell pair was theoretically simulated by boundary element method. Both the experimental observations and numerical simulations showed that, by increasing the acoustic pressure or reducing the bubble-cell distance, intensified deformation could be induced in the cellular membrane, which could result in enhanced intracellular delivery and cytoskeleton disassembly. The current results suggest that more tailored therapeutic strategies could be designed for ultrasound gene/drug delivery by adopting optimal bubble-cell distances and/or better controlling incident acoustic energy.