The atypical isoforms of protein kinase C (aPKCs) play an important role in insulin signaling and are involved in insulin stimulated glucose uptake in different cell systems. On the other hand, aPKCs also are able to negatively regulate important proteins for insulin signaling, like phosphatidyl-inositol-3-kinase (PI3K) and protein kinase B/Akt. To find aPKC-interacting proteins that may promote positive or negative activities of aPKCs, a yeast-2-hybrid-screen was performed. Partitioning-defective-protein 6 (Par6) was detected in human cDNA libraries of different adult insulin sensitive tissues. Although Par6 is known as an aPKC-interacting protein during development, no role for Par6 in insulin signaling has been reported so far. We therefore studied the effects of Par6 overexpression in C2C12 murine myoblasts. In these cells, Par6 associated constitutively with endogenous aPKCs, and the expression level as well as the activity of aPKCs were increased. Insulin-dependent association of the p85 subunit of PI3K with insulin-receptor-substrate1 (IRS1) was hampered and the phosphorylation of Akt/glycogen-synthase-kinase-3/ was significantly impaired after stimulation with insulin or with platelet-derived-growth-factor. Consequently, insulin-dependent glycogen synthesis was down-regulated (1.44 vs. 2.24 fold, P < 0.01). We therefore suggest that Par6 acts as a negative regulator of the insulin signal.