Objective Cell mechanics plays an important role in regulating cellular physiological and pathological processes and investigating cell mechanics is meaningful for revealing the underlying mechanisms guiding life activities. The advent of atomic force microscopy (AFM) provides a novel powerful tool for single-cell studies and AFM-based indentation assay has become an important method for characterizing cell mechanics in the field of life sciences, yielding numerous new insights into single-cell behaviors. However, current studies of cell mechanics by AFM are commonly performed in static environment, while cancerous cells are in flow environment of vascular fluids during tumor metastasis, and thus the results obtained in static environment cannot completely reflect the real behaviors of cancerous cells in fluidic condition. Particularly, so far knowledge of the mechanical mechanisms guiding the interactions between fluidic microenvironment and cancerous cells in the process of tumor metastasis is still limited. Here, based on AFM, a method allowing measuring the mechanical properties of single cells in fluidic environments is developed.Methods A fluidic cell culture medium device was established on a petri dish with openings on both sides with the use of an injection pump and an extraction pump. The fluidic cell culture medium device was integrated with AFM to measure the mechanical properties of cells in fluidic environments. The fluidic cell culture medium device was also integrated with inverted optical microscope which had a heating plate to observe the growth states of cells in fluidic environments. MCF-7 cells (human breast cancer cell) and HGC-27 cells (human gastric undifferentiated carcinoma cell) were used for the experiments. The fluidic cell culture medium device was used to grow cells to examine the effects of medium flow rate and flow time on cell proliferation and cell mechanics. Under the guidance of optical microscopy, AFM probe was moved to cells grown in static culture medium or fluidic culture medium to perform indentation assay to record force curves, and then Hertz-Sneddon model was applied to analyze the force curves to obtain the Young’s modulus of cells. Calcein fluorescein was used to stain live cells and PI fluorescein was used to stain dead cells. The cytoskeleton changes of MCF-7 cells after growth in fluidic culture medium were observed by confocal fluorescence microscopy.Results The effects of fluidic culture medium on the growth of cells were firstly analyzed. Experimental results on cell growth show that cell culture medium fluidic environment could better promote cell growth and proliferation compared with cell culture medium static environment. Then the mechanical properties of cells grown in static culture medium and fluidic culture medium were measured respectively. Experimental results show that, when the growth condition of cancerous cells changed from static to fluidic, the Young’s modulus of cancerous cells decreased significantly and cytoskeletons reorganized, indicating the influence of fluidic environment on the mechanics of cancerous cells.Conclusion Combining AFM with fluidic control techniques allows detecting the mechanical properties of single cells grown in fluidic environments, providing a novel way to investigate the mechanical cues involved in the interactions between fluidic microenvironments and cancerous cells during tumor metastasis.