The mechanisms regulating neutrophil transmigration of vascular endothelium are not fully elucidated, but involve neutrophil firm attachment and passage through endothelial cell-cell junctions. The goal of this study was to characterize the tangential forces exerted by neutrophils during transendothelial migration at cell-cell junctions using an in vitro laminar shear flow model in which confluent activated endothelium is grown on a microfabricated pillar substrate. The tangential forces are deduced from the measurement of pillar deflection beneath the endothelial cell-cell junction as neutrophils transmigrate. The force diagram displays an initial force increase, which coincides with neutrophil penetration into the intercellular space and formation of a gap in VE-cadherin staining. This is followed by a rapid and large increase of traction forces exerted by endothelial cells on the substrate in response to the transmigration process and the disruption of cell-cell contacts. The average maximum force exerted by an actively transmigrating neutrophil is three times higher than the force generated by an adherent neutrophil that does not transmigrate. Furthermore, we show that substrate rigidity can modify the mechanical forces induced by the transmigration of a neutrophil through the endothelium. Our data suggest that the force induced by neutrophil transmigration plays a key role in the disruption of endothelial adherens junctions.