OBJECTIEVE-Protein kinase C (PKC) theta activation is associated with insulin resistance and obesity, but the underlying mechanisms have not been fully elucidated. Impairment of insulin-mediated vasoreactivity in muscle contributes to insulin resistance, but it is unknown whether PKC theta is involved. In this study, we investigated whether PKC theta activation impairs insulin-mediated vasoreactivity and insulin signaling in muscle resistance arteries. RESEARCH DESIGN AND METHODS-Vasoreactivity of isolated resistance arteries of mouse gracilis muscles to insulin (0.02-20 nmol/l) was studied in a pressure myograph with or without PKC theta activation by palmitic acid (PA) (100 mu mol/l). RESULTS-In the absence of PKC theta activation, insulin did not alter arterial diameter, which was caused by a balance of nitric oxide-dependent vasodilator and endothelin-dependent vasoconstrictor effects. Using three-dimensional microscopy and Western blotting of muscle resistance arteries, we found that PKC theta is abundantly expressed in endothelium of muscle resistance arteries of both mice and humans and is activated by pathophysiological. levels of PA, as indicated by phosphorylation at Thr(538) in mouse resistance arteries. In the presence of PA, insulin induced vasoconstriction (21 +/- 6% at 2 nmol/l insulin), which was abolished by pharmacological or genetic inactivation of PKC theta. Analysis of intracellular signaling in muscle resistance arteries showed that PKC theta activation reduced insulin-mediated Akt phosphorylation (Ser(473)) and increased extracellular signal-related kinase (ERK) 1/2 phosphorylation. Inhibition of PKC theta restored insulin-mediated vasoreactivity and insulin-mediated activation of Akt and ERK1/2 in the presence of PA. CONCLUSIONS-PKC theta activation induces insulin-mediated vasoconstriction by inhibition of Akt and stimulation of ERK1/2 in muscle resistance arteries. This provides a new mechanism linking PKC theta activation to insulin resistance.