The dynamic physical microenvironment of bone affects the activity of osteoblast cells, yet little is known about how osteoblast mechanotransduction depends on different features of a dynamic stimulus. Here we investigated the effect of physiologically relevant oscillatory flow shear stress on the calcium mobility in osteoblast cells within a microfluidic platform that mimics the confined environment of bone matrix. We characterized the spatiotemporal evolution of intracellular calcium 'flickers', an important signature of cell activation, in response to steady, pulsatile, and oscillatory shear stress. We found that oscillatory flow induces surprisingly higher flicker activity than other flow types. We could further attribute this phenomenon to the opening of a stretch activated ion channel, namely TRPM7. We also found that localization of TRPM7 within the cholesterol-enriched lipid raft domains of plasma membranes is essential for its activity. Collectively our findings elucidated a candidate mechanism for the flow mediated stimulation of osteoblast cells. They therefore have implications towards unveiling various facets of bone formation and remodelling in healthy and diseased conditions, including bone-metastasis of various cancer types, diabetes, and inflammatory autoimmune diseases. © The Royal Society of Chemistry 2014.