The advent of atomic force microscopy (AFM) provides a powerful tool for investigating the behaviors of single living cells under near physiological conditions. Besides acquiring the images of cellular ultra-microstructures with nanometer resolution, the most remarkable advances are achieved on the use of AFM indenting technique to quantify the mechanical properties of single living cells. By indenting single living cells with AFM tip, we can obtain the mechanical properties of cells and monitor their dynamic changes during the biological processes (e.g., after the stimulation of drugs). AFM indentation-based mechanical analysis of single cells provides a novel approach to characterize the behaviors of cells from the perspective of biomechanics, considerably complementing the traditional biological experimental methods. Now, AFM indentation technique has been widely used in the life sciences, yielding a large amount of novel information that is meaningful to our understanding of the underlying mechanisms that govern the cellular biological functions. Here, based on the authors’ own researches on AFM measurement of cellular mechanical properties, the principle and method of AFM indentation technique was presented, the recent progress of measuring the cellular mechanical properties using AFM was summarized, and the challenges of AFM single-cell nanomechanical analysis were discussed.