The insulin receptor phosphorylates insulin receptor substrate (IRS) proteins on multiple tyrosine residues that act as docking sites to recruit a number of downstream signaling molecules. Here we show that IRS3 is localized both at the plasma membrane and in the nucleus. Interestingly, the nuclear localization of the protein is restricted to specific regions involved in mRNA processing and known as speckles. By using different truncated versions of the protein, we demonstrate that the pleckstrin homology (PH) domain is involved in IRS3 localization at the level of both plasma membrane and nucleus. To our knowledge this is the first report of a PH domain responsible for a nuclear targeting of the host protein. By site-directed mutagenesis, we identify residues within the PH domain critical for proper localization of IRS3. Mutations within the PH domain preventing IRS3 intracellular localization result in an inhibition of IRS3-induced glucose uptake. We conclude that the PH domain is required for IRS3 intracellular localization and, furthermore, that it has a key role in metabolic functions of IRS3. In particular, our data suggest that IRS3 intracellular localization at the plasma membrane and in the nucleus is the result of two different cooperative mechanisms both involving the PH domain.