Objective To investigate the specific roles of gap junction and ATP in mechanical stimulation induced calcium transfer in osteoblasts. Methods The isolated osteoblastic pattern without gap junctions was established by using the micropatterning method. Then fluid shear stress was applied on cells using the flow chamber to observe and analyze the characteristic parameters of calcium response. Results Multiple calcium response still occurred in osteoblastic pattern without gap junction, but the response time to the first responsive peak was much longer than that with gap junction. When the intracellular and extracellular calcium ions were removed, only 40% cells responded to the mechanical stimulation, with single peak and multiple peaks accounting for 50%, respectively. If ATP pathway was blocked, only 20% cells responded, most of which showed single peak. Conclusions ATP was the major pathway mediating intercellular calcium transfer, while the gap junction was not the necessary one.