Angiopoietin-2 (Ang2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS), in a vascular endothelial cell (VEC)-specific and Ang2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided co-culture model with VECs and VSMCs from Sprague Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang2 effects were suppressed by the MEGJ inhibitor, 18α-glycyrrhetic acid (18-GA), Tie2 siRNA or Cx43 siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang2 treatment, was antagonized by Cx43 siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.