Protein kinase C (PKC) induces endothelial dysfunction, which is an important pathological factor in cardiovascular diseases. This study aimed to evaluate the role of PKCβII on the endothelial dysfucntion related to mitochondrial activation.  Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on the endothelial dysfucntion were investigated in human umbilical vein endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1 and monocyte adhesion which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD) which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting CREB plays a key role of MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. On contrast, overexpression of PKCβII using AdPKCβII increased mitochondrial ROS, MnSOD, ICAM-1 and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression is inhibited by Mito-TEMPO, mitochondrial ROS scavenger, suggesting involvement of mitochondrial ROS in PKC-induced vascular inflammation.  Taken together, it is suggested that PKCβII plays an important role of PMA-induced endothelial dysfunction, and the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.