IRS-2 mediates insulin-induced glucose uptake in brown preadipocytes. Upon differentiation, basal IRS-3 expression increased concurrently with an enhancement in the IRS-3-associated phosphatidylinositol (PI) 3-kinase activity in the Triton-insoluble fraction in wild-type and IRS-2-deficient brown adipocytes stimulated with insulin. Moreover, insulin induced protein kinase B (Akt) and protein kinase C (PKC) zeta phosphorylation in both kinds of cells. More importantly, insulin induced glucose uptake in differentiated IRS-2-deficient brown adipocytes in a wortmannin-dependent manner. However, while insulin induced Akt phosphorylation occurred mainly in the cytosolic fraction, PKC zeta activation was constrained to the Triton-insoluble fraction. The reduction of IRS-3 expression by siRNA inhibited insulin-induced glucose uptake and also PKC zeta activation in differentiated IRS-2(-/-) brown adipocytes. In addition, inhibition of PKC zeta totally blunted insulin-induced glucose uptake in those cells. Our results provide evidences suggesting that IRS-3/PI 3-kinase/PKC zeta signaling is the main responsible for the insulin-induced glucose uptake observed upon differentiation of brown adipocytes lacking IRS-2.