Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS). Alterations in actin cytoskeleton are shown to be crucial for NF-B regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin, in mediating cytoskeletal alterations essential for NF-B activation and EC inflammation. The present study, describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammmatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-B activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion also inhibited RelA/p65 phosphorylation at Ser⁵³⁶, a critical event conferring transcriptional competency to the bound NF-B. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 from IBα by blocking its phosphorylation/degradation. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNFα-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.