1, 25-dihydroxyvitamin D3 (1, 25 (OH)2 D3) and mechanical stimuli in physiological environment contributes greatly to osteoporosis pathogenesis. Wide investigations have been conducted on how 1, 25-dihydroxyvitamin D3 and mechanical stimuli separately impact osteoblasts. This study reports the collective influences of 1, 25-dihydroxyvitamin D3 and flow shear stress (FSS) on biological functions of osteoblasts. 1, 25 (OH)2 D3 were prepared in various kinds of concentrations (0, 1, 10, 100nmmol/L), while physiological fluid shear stress (12dynes/cm2) was produced by using a parallel-plate fluid flow system. 1, 25 (OH)2 D3 affects the responses of ROBs to FSS, including the inhibition of NO release and cell proliferation as well as the promotion of PGE2 release and cell differentiation. These findings provide a possible mechanism by which 1, 25(OH)2 D3 influences osteoblasts’ responses to FSS, thus most probably providing guidance for the selection of 1, 25(OH)2 D3 concentration and mechanical loading in order to produce functional bone tissues in vitro.