We tested the hypothesis that elevated membrane cholesterol of leukocytes (associated with elevated plasma cholesterol levels due to hypercholesterolemia) influences their sensitivity to shear. For this purpose, we assessed the shear stress regulation of pseudopod activity of human neutrophils after membrane cholesterol‐modifying treatments and of leukocytes from hypercholesterolemic mice. Our results show that, compared to naive cells, migrating human neutrophils with enhanced membrane cholesterol exhibited impaired pseudopod retraction during micropipette shear (τmax~2 dyn/cm2, 2 min). Moreover, significantly elevated (>4‐fold) blood cholesterol levels in LDL receptor‐deficient (LDLR−/−) mice fed a high fat diet (WD) for 2 wks compared to those in LDLR−/− (ND) or wild‐type (WT) mice on regular chow correlated with an impaired shear response; suspended neutrophils from WD did not exhibit significant reductions in pseudopod activity after exposure to cone‐plate shear (5 dyn/cm2, 5 min). Although plasma cholesterol levels in ND were ~2‐fold higher than those in WT, neutrophils from ND still responded to shear with reduced pseudopod activity. Combined, these results suggest that elevated membrane cholesterol impairs the leukocyte shear response, a perturbation that may contribute to chronic leukocyte activity in hypercholesterolemia. Funded by the NIH, the UKY Research Support Fund and the AHA.