The purpose of the present study is to investigate the changes of Rho kinase activity and its role in biphasic response of vascular reactivity and calcium sensitivity after hemorrhagic shock. The vascular reactivity and calcium sensitivity of superior mesenteric artery (SMA) from hemorrhagic shock rats were determined via observing the contraction initiated by norepinephrine (NE) and Ca under depolarizing conditions (120 mmol/L K) with isolated organ perfusion system. At same time, Rho kinase activity in mesenteric artery was measured, and the effects of Rho kinase activity-regulating agents, angiotensin II (Ang-II), insulin, and Y-27632, on vascular reactivity and calcium sensitivity were also observed. The results indicated that the vascular reactivity and calcium sensitivity were increased at early shock (immediate and 30 min after shock) and decreased at late shock (1 and 2 h after shock). The maximal contractions of NE and Ca were significantly increased (P < 0.05 or P < 0.01) at early shock. But they were significantly decreased at late shock (P < 0.05 or P < 0.01). Rho kinase activity was significantly increased at early shock (immediate after shock) (P < 0.05) but significantly decreased at 1 and 2 h after shock (P < 0.05 or P < 0.01). It was positively correlated with the changes of vascular reactivity and calcium sensitivity. Insulin decreased the increased contractile response of SMA to NE and Caat early shock (P < 0.05 or P < 0.01). Angiotensin II increased the decreased contractile response of SMA to NE and Ca at 2-h shock (P < 0.05 or P < 0.01); Y-27632, Rho kinase-specific antagonist, decreased the contractile response of SMA to NE and Ca at 2-h shock, and abolished Ang-II induced the increase of vascular reactivity and calcium sensitivity. The results suggest that Rho kinase may be involved in the biphasic change of vascular reactivity and calcium sensitivity after hemorrhagic shock. Rho kinase may regulate vascular reactivity through the regulation of calcium sensitivity. Rho kinase-regulating agents may have some beneficial effects on shock-induced vascular hyporeactivity.