Many cells respond to fluid shear stress but in a cell-type specific fashion. Fluid shear applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are, however, still unknown. We hypothesize that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased constitutive activity of G-protein coupled receptor (GPCR). Inhibition of GPCR constitutive activity by inverse agonists abolished shear-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells with lack of FPR form few pseudopods and no detectable response to fluid shear, while expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR siRNA-transfected differentiated HL60 cells exhibited no response to fluid shear. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection.