Cover Story：Objective Cell mechanics plays an important role in cellular physiological and pathological processes and is closely related to the health states of living organisms. Investigating cell mechanics significantly benefits revealing the underlying mechanisms guiding life activities. The advent of atomic force microscope (AFM) provides a novel instrument for single-cell assay. AFM is able to not only visualize the morphology of singe living cells under aqueous conditions with high resolution, but also quantitatively measure the mechanical properties of cells. Utilizing AFM to investigate the mechanics of individual cells has achieved great success in the past decades, which provides numerous new insights into cellular physiological and pathological processes and has become an important tool in the field of life sciences. However, due to the fact that AFM probe itself is unable to perform drug delivery, so far it is still challenging for the simultaneous measurements of cell mechanics by AFM in response to the stimulation of ultra-trace drug. Here, by combining micropipette and AFM, a method allowing single-cell precise drug delivery and simultaneous measurements of cell mechanics is presented. Methods The micropipette-based single-cell microinjection system was built on an inverted fluorescent microscope by using a 3D manipulator, a micropump, a syringe, a PTFE tube and a micropipette. The micropipette was obtained from the glass capillary by using the micropipette puller. NIH 3T3 cells (mouse embryonic fibroblast), HEK 293 cells (human embryonic kidney cell) and MCF-7 cells (human breast cancer cell) were used for the experiments. Under the guidance of optical microscopy, staining reagents or drug molecules were delivered to individual cells, and then AFM probe was moved to the targeted cells to obtain force curves. Cellular Young’s modulus was calculated from the force curves by applying Hertz-Sneddon model. Results The effects of the pore size of micropipette tip on cell injection were analyzed firstly, and the results showed that larger pore size tip (the outer diameter of the tip was larger than 1 μm) could cause obvious mechanical damage to the cell. Then blue ink or PI staining solution was injected to single cells by micropipette under the guidance of optical microscopy, and the recorded optical/fluorescent images after injection clearly showed that the targeted cells were successfully injected. Finally, micropipette was integrated with AFM to measure the Young’s modulus changes of single cells after the treatment of chemotherapeutic drug (cytarabine), and the results showed that stimulation of cytarabine could cause the changes of cellular mechanical properties. Conclusion Combining micropipette and AFM enables applying precise chemical stimulation to a single cell while simultaneously measuring cellular mechanical properties after chemical stimulation, providing a novel idea for single-cell mechanical analysis in response to ultra-trace drugs.