Bone cells are adaptive to surrounding mechanical conditions. Osteoblasts, one of bone cells, have been reported to be sensible to mechanical stimulation and change the generated bone mass. Activation of the signaling molecules in relation to the initial mechanoreception appears to be mediated though changes in the cytoskeleton. In this study, we propose a method to visualize cytoskeletal actin fibers in a living osteoblast under applied shear deformation and measure the local deformation of actin fibers. Transfection of MC3T3-E1, which is an osteoblast-like cell line, with GFP-actin was performed using a transfection reagent. Shear deformation was applied to the cell using a micropipette. As a result of the experiment, it is shown to be able to recognize the specific points such as points crossed by the actin fibers and obtain the local deformation vectors of the actin fiber network at the specific points in the cell. The present method contributes to not only obtaining the deformation distribution of actin fibers in cells but also understanding the mechanotransduction mechanisms.