Resuscitation from hemorrhagic shock induces endothelial dysfunction and activates inflammatory cascades leading to organ damage. Following restoration of blood flow to ischemic vascular beds, leukocyte–endothelium interactions leading to leukocyte infiltration into the vascular wall occur very early due, in part, to the loss of endothelium-derived nitric oxide (NO). The mechanism by which ischemia–reperfusion injury impairs endothelium-derived NO is not completely understood. We hypothesized that inhibition of Rho-kinase could exert beneficial effects following hemorrhagic shock by preserving endothelial function and attenuating leukocyte trafficking in the microcirculation. Using intravital microscopy, we found that resuscitation from hemorrhage acutely increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. Treatment of mice with the Rho-kinase inhibitor fasudil, markedly attenuated leukocyte–endothelium interaction in response to hemorrhage/reinfusion. The beneficial effect of fasudil was not observed in endothelial nitric oxide synthase (eNOS)−/− mice. In conclusion, inhibition of Rho-kinase prevents inflammatory leukocyte trafficking in the microcirculation via an eNOS-dependent mechanism. Our data support a role for Rho-kinase inhibitors in the treatment of ischemia–reperfusion injury.