Nucleophosmin (NPM) is a multifunctional phosphoprotein, being involved in ribosome assembly, pre-ribosomal RNA processing, DNA duplication, nucleocytoplasmic protein trafficking, and centrosome duplication. NPM is phosphorylated by several kinases, including nuclear kinase II, casein kinase 2, Polo-like kinase 1 and cyclin-dependent kinases (CDK1 and 2), and these phosphorylations modulate the activity and function of NPM. We have previously identified Thr(199) as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2-mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr(199). We found that the phospho-Thr(199) NPM localized to dynamic sub-nuclear structures known as nuclear speckles, which are believed to be the sites of storage and/or assembly of pre-mRNA splicing factors. Phosphorylation on Thr(199) by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA-binding activity of NPM. Moreover, phospho-Thr(199) NPM, but not unphosphorylated NPM, effectively represses pre-mRNA splicing. These findings indicate the involvement of NPM in the regulation of pre-mRNA processing, and its activity is controlled by CDK2-mediated phosphorylation on Thr(199).