Insulin-stimulated glucose transport is impaired in the early phases of type 2 diabetes mellitus. Studies in rodent cells suggest that atypical PKC (aPKC) isoforms (ζ, λ, and ι) and PKB, and their upstream activators, PI3K and 3-phosphoinositide-dependent protein kinase-1 (PDK-1), play important roles in insulin-stimulated glucose transport. However, there is no information on requirements for aPKCs, PKB, or PDK-1 during insulin action in human cell types. Presently, by using preadipocyte-derived adipocytes, we were able to employ adenoviral gene transfer methods to critically examine these requirements in a human cell type. These adipocytes were found to contain PKC-ζ, rather than PKC-λ/ι, as their major aPKC. Expression of kinase-inactive forms of PDK-1, PKC-ζ, and PKC-λ (which functions interchangeably with PKC-ζ) as well as chemical inhibitors of PI 3-kinase and PKC-ζ/λ, wortmannin and the cell-permeable myristoylated PKC-ζ pseudosubstrate, respectively, effectively inhibited insulin-stimulated glucose transport. In contrast, expression of a kinase-inactive, activation-resistant, triple alanine mutant form of PKB-α had little or no effect, and expression of wild-type and constitutively active PKC-ζ or PKC-λ increased glucose transport. Our findings provide convincing evidence that aPKCs and upstream activators, PI 3-kinase and PDK-1, play important roles in insulin-stimulated glucose transport in preadipocyte-derived human adipocytes.