Rho-kinase signaling regulates inflammatory cell migration and chemokine production. We therefore investigated mechanisms of Rho-kinase dependent inflammation in lipopolysaccharide (LPS)-induced renal failure. C57/BL6 mice received intraperitoneal LPS with or without daily treatment with specific Rho-kinase inhibitors (Y-27632 or HA-1077, 5 mg/kg). Rho-kinase inhibitors were applied in a preventive (12h or 1h prior to LPS) or a therapeutic (6h after LPS) scheme. Both protected renal function and decreased tubular injury in LPS treated mice. Enhanced Rho-kinase activity was inhibited by HA-1077 in capillary endothelial cells, inflammatory cells and tubuli by analysis of Rho-kinase substrate phosphorylation. Early neutrophil influx was reduced by HA-1077 without reduction of the proinflammatory cytokine TNF. In contrast, HA-1077 decreased influx of monocytes/macrophages coinciding with reduced expression of the NFB regulated chemokines CCL5 and CCL2. We therefore examined NFB signal transduction and found that NFB p65 phosphorylation and nuclear translocation were reduced by Rho-kinase inhibition. IB degradation was not altered during the first 6h but reduced by HA-1077 at later time points. NFB p50 deficient mice were similarly protected from renal injury by Rho-kinase inhibition further supporting the prominent role for p65 in Rho-kinase inhibition. Together, these data suggest that Rho-kinase inhibition by preventive or therapeutic treatment effectively reduced endotoxic kidney injury in part by attenuation of NFB p65 activation.