A single nucleotide polymorphism in the partitioning defective protein-6alpha (Par6alpha) promoter is coupled with lower Par6alpha expression and better insulin sensitivity, whereas overexpression of Par6alpha in C2C12 myoblasts inhibits insulin-induced protein kinase B/Akt1 activation and glycogen synthesis. Here we show that a direct interaction of Par6alpha with atypical protein kinase C (aPKC) is crucial for this inhibition. A DeltaPB1-Par6alpha deletion mutant that does not interact with aPKC neither increased aPKC activity nor interfered with insulin-induced Akt1 activation in C2C12 cells. Further, T34 phosphorylation of Akt1 through aPKC is important for inhibition of Akt1. When Par6alpha was overexpressed, activation of wild-type Akt1 (-59.3%; p=0.049), but not T34A-Akt1 (+2.9%, p=0.41) was reduced after insulin stimulation. The resistance of T34A-Akt1 to Par6alpha/aPKC-mediated inhibition was also reflected by reconstitution of insulin-induced glycogen synthesis. In summary, Par6alpha-mediated inhibition of insulin-dependent glycogen synthesis in C2C12 cells depends on the direct interaction of Par6alpha with aPKC and on aPKC-mediated T34 phosphorylation of Akt1.