Utilizing cell culture medium to grow cells in vitro has been widely studied in the past decades and has been recognized as an acknowledged way for investigating cell activities. However, due to the lack of adequate observation tools, the detailed mechanisms regulating cell growth in cell culture medium are still not fully understood. In this work, atomic force microscopy (AFM), a powerful tool for observing native biological systems under near-physiological conditions with high resolution, was applied to reveal the nanogranular surfaces formed in cell culture medium in situ for promoting cell growth. First, AFM imaging of glass slides (glass slides were previously incubated in cell culture medium) in aqueous environment clearly visualized the cell culture medium-forming nanogranular surfaces on glass slides. By altering the incubation time of glass slides in cell culture medium, the dynamic formation of nanogranular surfaces was remarkably observed. Next, fluorescent labeling experiments of the cell culture medium-treated glass slides showed that bovine serum proteins were contained in the nanogranular surfaces. Further, the adhesive interactions between cells and nanogranular surfaces probed by AFM force spectroscopy and the cell growth experiments showed that cell culture medium-forming nanogranular surfaces promote cell attachment and growth. The study provides novel insights into nanotopography-regulated molecular mechanisms in cell growth and demonstrates the outstanding capabilities of AFM in addressing biological issues with unprecedented spatial resolution under aqueous conditions, which will have potential impacts on the studies of cell behaviors and cell functions. IEEE