Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P₃ second messenger. PtdIns(3,4,5)P₃ can be broken down to PtdIns(3,4)P₂ through the action of the SHIPs (Src-homology-2-domain- containing inositol phosphatases). As PtdIns(3,4)P₂ levels peak after those of PtdIns(3,4,5)P₃, it has been proposed that PtdIns(3,4)P₂ controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P₂ through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P₂. These homozygous TAPP1^R211L/R211LTAPP2 ^R218L/R218L double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1 ^R211L/R211LTAPP2^R218L/R218L knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P ₃ and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adaptors to PtdIns(3,4)P₂ function as negative regulators of the insulin and PI3K signalling pathways.