Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and impairs further insulin induction. Like glucose transport, protein kinase C (PKC)-α and -β are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6_PED). PKC-ζ features no basal change but completely loses insulin sensitivity in L6_PED. In these cells, blockage of PKC-α and -β additively returns 2-deoxy-d-glucose (2-DG) uptake to the levels of cells expressing only endogenous PED (L6_WT). Blockage of PKC-α and -β also restores insulin activation of PKC-ζ in L6_PED cells, with that of PKC-α sixfold more effective than PKC-β. Similar effects on 2-DG uptake and PKC-ζ were also achieved by 50-fold overexpression of PKC-ζ in L6_PED. In L6_WT, fivefold overexpression of PKC-α or -β increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor substrate phosphorylation. In these cells, overexpression of PKC-α blocks insulin induction of PKC-ζ activity. PKC-β is 10-fold less effective than PKC-α in inhibiting PKC-ζ stimulation. Expression of the dominant-negative K²⁸¹→W PKC-ζ mutant simultaneously inhibits insulin activation of PKC-ζ and 2-DG uptake in the L6_WT cells. We conclude that activation of classic PKCs, mainly PKC-α, inhibits PKC-ζ and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells.