Biochemical signals related to a mechanosensory mechanism by which cells sense mechanical stimuli have been gradually clarified by biological approaches such as blocking specific signaling pathways; however, mechanical signals such as deformation/strain, which is transduced into biochemical signals through this mechanism, particularly at the cellular structural component level in a single cell, have not yet been clearly understood. This in vitro study focuses on an intracellular calcium signaling response to an applied localized deformation in a single osteoblast-like MC3T3-E1 cell, and observed localized deformation of a cell membrane and the calcium ion flux from an extracellular medium. The localized deformation was applied to a cell by indenting a microsphere onto the cell membrane using a glass microneedle. The cellular calcium signaling response and cell membrane deformation were simultaneously observed using fluorescent dyes in a vertical section under a confocal laser-scanning microscope with high spatial and temporal resolutions. Our results observed in the vertical section showed that the initiation point of the calcium ion flux is collocated at the displaced microsphere around which stretch membrane deformation was observed.