Phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol-4,5-biphosphate (PIP2) are two well-known membrane bound polyphosphoinositides. Diabetes is associated with impaired glucose metabolism. Using a 3T3L1 adipocyte cell model, this study investigated the role of PIP3 and PIP2 on insulin stimulated glucose metabolism in high glucose (HG) treated cells. Exogenous PIP3 supplementation (1, 5, or 10 nM) increased the phosphorylation of AKT and PKCζ/λ, which in turn upregulated GLUT4 total protein expression as well as its surface expression, glucose uptake, and glucose utilization in cells exposed to HG (25 mM); however, PIP2 had no effect. Comparative signal silencing studies with antisense AKT2 and antisense PKCζ revealed that phosphorylation of PKCζ/λ is more effective in PIP3 mediated GLUT4 activation and glucose utilization than in AKT phosphorylation. Supplementation with PIP3 in combination with insulin enhanced glucose uptake and glucose utilization compared to PIP2 with insulin, or insulin alone, in HG-treated adipocytes. This suggests that a decrease in cellular PIP3 levels may cause impaired insulin sensitivity in diabetes. PIP3 supplementation also prevented HG-induced MCP-1 and resistin secretion and lowered adiponectin levels. This study for the first time demonstrates that PIP3 but not PIP2 plays an important role in GLUT4 upregulation and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation. Whether PIP3 levels in blood can be used as a biomarker of insulin resistance in diabetes needs further investigation.