We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock, and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser262 and gap-junctional communication in VSMCs. Activation of RhoA with U46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junctions independent. Gap junctions between VSMCs, gap-junctional communication and Cx43 phosphorylation at Ser262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process.