Nucleostemin (NS) is a nucleolar protein involved in the regulation of cell proliferation. Both overexpression and knock-down ofNSincrease the activity of the tumor suppressor protein p53, resulting in cell cycle arrest. In addition, NS regulates processing of pre-rRNA and consequently the level of total protein synthesis. Here, we describe a previously uncharacterized function of NS in the maintenance of the tripartite nucleolar structure as well as the integrity of small nucleolar ribonucleoproteins (snoRNPs). NS is also necessary to maintain the telomerase complex which shares common protein subunits with the H/ ACA box snoRNPs. First, immunofluorescence microscopy and electron microscopy demonstrated that knockdown of NS disorganized the nucleolar architecture, in particular, the dense fibrillar component where snoRNPs are localized. Second, gel filtration chromatography and immunoprecipitation indicated that NS depletion leads to dissociation of the components of snoRNPs and the telomerase complex. Third, NS depletion reduced both telomerase activity and the cellular level of pseudouridine, an H/ACA snoRNP-mediated modification of rRNA and other RNAs that are important for their folding and stability. These morphological, biochemical and functional studies demonstrate thatNSplays an important role to maintain nucleolar structure and function on a more fundamental level than previously thought.