Impaired vascular regulation as a result of endothelial dysfunction is a major determinant of sepsis-associated liver failure. We have previously shown that inhibition of one signaling molecule rho-kinase (ROCK) predominantly restores endothelial function impaired by endotoxin lipopolysaccharide. In the present study, we determined a ligand that activates ROCK mediated endothelial dysfunction. A potential candidate is sonic hedgehog (SHH) as it activates RhoA/ROCK non-canonically. More relevantly, T-lymphocytes that undergo massive apoptosis in septic patients release SHH in vesicles called microparticles (MPs). However, the role and mechanisms of SHH+MPs in endothelial dysfunction is not completely delineated. In this study, MPs were derived by inducing apoptosis of human T-lymphoblastoid cell line and contained SHH (80%). These SHH+MPs induced stress fibers, hyperpermeability (+71.5%), mitochondrial depolarization (-61.5%), increased oxidative stress (+242%) and decreased nitric oxide production (-46%) in HMECs and impaired endothelium-dependent vasodilation (-61.82%) in ex-vivo porcine mesenteric rings, by activating RhoA/ROCK pathway. SHH agonist also caused the same endothelial dysfunction as SHH+MPs. Inhibition of the SHH pathway or ROCK before addition of MPs abrogated endothelial dysfunction, indicating that activation of ROCK through the SHH pathway is required for MP-induced endothelial dysfunction. In summary, this study elucidated a novel mechanism in which apoptotic T-cell derived MPs containing SHH induce endothelial dysfunction via RhoA/ROCK pathway and thereby can contribute to vascular dysfunction of liver during sepsis