Objective To investigate the E-selectin mediated-calcium response of neutrophils under flow fields. Methods A parallel-plate flow chamber combined with a fluorescence microscope was used to observe the adhesion and subsequent calcium response of neutrophils on E-selectin at different concentrations under fluid shear stress (FSS) of 0-600 mPa. Results E-selectin could capture neutrophils from the flow to the chamber substrate and induce further intracellular calcium flux of firmly adhered cells. The arrested cell and activation ratio increased gradually as the concentration of E-selectin increased. Only immobilized E-selectin could conduct the external force-signal to trigger the calcium response of neutrophils effectively. By increasing FSS, not only was the activation ratio increased from 23% to 70%, but the calcium response intensity also increased from 0.92 to 1.45, while the delay time from cell adhesion to calcium response was greatly reduced from 70 s to 27 s. Conclusions FSS can modulate the calcium response of neutrophils in cooperation with E-selectin, and it positively regulates the activation rate and level of calcium response. This study may deepen the understanding of the immune response of leukocytes under a hemodynamic environment.