IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.