Mechanical cues play a crucial role in regulating physiological and pathological processes, and atomic force microscopy (AFM) has become an important and standard tool for measuring the mechanical properties of single cells. In particular, providing a capability to manipulate cells in a three-dimensional (3D) space benefits enhancing the applications of AFM measurements in cell biology. Here, we present the complementary integration of AFM and micropipette micromanipulation, which allows precise 3D manipulations and nanomechanical measurements of single living cells. A micropipette micromanipulation system under the guidance of optical microscopy was established to isolate single living cells, and polydimethylsiloxane (PDMS) micropillar substrates were used to physically immobilize the isolated living cells for downstream AFM detection. The viscoelastic properties (Young's modulus, relaxation time, viscosity) of cells were quantitatively measured by AFM-based indentation assay. The effectiveness of micropipette-assisted AFM in single-cell analysis was confirmed on both living animal suspended cells and living animal adherent cells, showing dramatic changes in cell mechanics in different states and revealing the dynamics of single cells grown on micropillar arrays. The study demonstrates the great potential of a micropipette to aid AFM in single-cell manipulations for better accessing the mechanical cues involved in cellular processes, which will allow additional studies of single-cell mechanics and will benefit the field of mechanobiology.