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 Thr199 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 Thr199. We found that the phospho‐Thr199 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 Thr199 by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA‐binding activity of NPM. Moreover, phospho‐Thr199 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 Thr199.