Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues is believed to play various regulatory roles in IRS protein function. However, studies of serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 at these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing the level of IRS2 protein degradation. Significantly, we show that IRS2 phosphorylation is cell cycle-regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation.