Based on recent studies showing that PLCγ associates to insulin receptor, we investigated its role in insulin stimulation of glucose transport in brown adipocytes. Insulin stimulation induced rapid PLCγ association to phosphorylated insulin receptor, and activation of PLCγ, as assessed by the mobilization of Ca²⁺ from intracellular stores and by the production of the second messenger DAG. Both events are dependent on activation of PI3-kinase. Inhibition of PLCγ activity either with the chemical compound U73122 or with an inhibitor peptide precluded insulin stimulation of glucose uptake, GLUT4 translocation, and actin reorganization, as wortmannin did. In contrast, the inactive analog U73343 did not have an inhibitory effect. Furthermore, translocation of GLUT4–GFP in response to insulin was completely abolished by cotransfection with a PLCγ-inactive mutant in HeLa cells, a cell model sensitive to insulin that express PLCγ. U73122 did not affect PI3-kinase nor Akt activation, but impaired PKCζ activation by insulin, as wortmannin did. PLC activity renders two products, IP₃ and DAG, and DAG can be metabolized to PA by the action of DAG-kinase. Using the compound R54494, a DAG-kinase inhibitor, insulin-induced PKCζ activation was also suppressed, this activity being restored by addition of PA. In summary, these data indicate that PLCγ, activated at least partially by PI3-kinase, is a link between insulin receptor and PKCζ through the production of PA and could mediate insulin-induced glucose uptake and GLUT4 translocation.