Insulin/insulin-like growth factor-1 (IGF-1)-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism and autophagy in various cells including hematopoietic cells. Although the early protein kinase activation cascade has intensively been studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed the high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells one hour after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 (Rho-GDI-2) and glutathione s-transferase-pi (GST-pi). Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, up-regulation of CLIC1 was not observed in one-dimensional Western blotting unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus, insulin induces the proteasome-dependent degradation of SRp20 as well as the sub-nuclear relocalization of CLIC1.