Objective To study mechanical responses from mesenchymal stem cells (MSCs) under different mechanical stimulus duration, by measuring its elastic modulus and characterizing its stress fibers. Methods High resolution images of MSCs cytoskeleton in vitro were acquired by using atomic force microscope (AFM) and laser scanning confocal microscope (LSCM). AFM cantilever with micro-bead attached probe was used to perform force-distance curve experiment on MSCs at the approaching time of 0.1，0.5, 1, 5,10 s, respectively. The elastic modulus of MSCs at 300 nm indentation depth were measured and compared. Results The rat MSCs cytoskeleton presented an intensely organized network structure. The elastic modulus of rat MSCs varied obviously for different mechanical stimulus duration. The median and quartile (QR) of MSCs elastic modulus were 10.02 (QR=9.66),1.94 (QR=7.71),3.63 (QR=19.33),17.15(QR=35.13), 23.52 kPa(QR=34.87), with probe approaching time at 0.1，0.5, 1, 5,10 s, respectively. The MSCs elastic modulus showed the tendency of increasing with stimulus duration increasing, except for the extremely short stimulus (0.1 s). Conclusions Unlike inorganic elastomer, rat MSCs possess complete and flexible mechanical load-bearing structure and can respond actively to a relatively longer mechanical stimulation, with an increase of elastic modulus. These results may provide basic data for further tissue engineering researches on mechanical regulation of MSCs behavior.