Extracellular matrix plays an important role in regulating the behaviors of cells, and utilizing matrix physics to control cell fate has been a promising way for cell and tissue engineering. However, the nanoscale situations taking place during the topography-regulated cell-matrix interactions are still not fully understood to the best of our knowledge. The invention of atomic force microscopy (AFM) provides a powerful tool to characterize the structures and properties of living biological systems under aqueous conditions with unprecedented spatial resolution. In this work, with the use of AFM, structural and mechanical dynamics of individual cells grown on micro-/nanotopographical surface were revealed. First, the microgroove patterned silicon substrates were fabricated by photolithography. Next, nanogranular topography was formed on microgroove substrates by cell culture medium protein deposition, which was visualized by in situ AFM imaging. The micro-/nanotopographical substrates were then used to grow two types of cells (3T3 cell or MCF-7 cell). AFM morphological imaging and mechanical measurements were applied to characterize the changes of cells grown on the micro-/nanotopographical substrates. The experimental results showed the significant alterations in cellular structures and cellular mechanics caused by micro-/nanotopography. The study provides a novel way based on AFM to unveil the native nanostructures and mechanical properties of cell-matrix interfaces with high spatial resolution in liquids, which will have potential impacts on the studies of topography-tuned cell behaviors.