Nucleostemin (NS) is a nucleolar protein expressed in stem and cancer cells. In combination with nuclear/nucleolar proteins, NS has been demonstrated to be involved in cell-cycle regulation and telomere maintenance. NS expression reflects the cell's proliferation state indicating that the cell is active in the cell cycle, whereas NS signals disappear upon differentiation. This study analyzes the spatio-temporal (nucleolar/nuclear localization during interphase and M-phase) NS remodeling in two distinct human mesenchymal stem cell (MSC) populations to discriminate the NS differences, if any, throughout their stem cell and differentiation states. Beside its prominent multilobular nucleolar localization in interphase cells, coexistence of NS with chromosome arms during mitosis was also observed. Disruption of mitotic microtubules induced dissociation of NS from the chromosome arms and scattered it into the cytoplasm. Compared to deciduous dental pulp MSCs, NS mRNA expression gradually decreased upon aging in umbilical cord stroma-derived MSCs as culture time increased. Following adipogenic differentiation of the latter, NS signals gradually disappeared in both dividing and non-dividing cells, even before the morphological and functional signs of adipogenic transformation appeared. Quantitative NS mRNA measurements showed that MSCs from two sources exhibit a strong nucleostemin expression similar to embryonic stem cells. In conclusion, apart from its novel chromosomal localization shown in this study, nucleolar NS can be considered as a marker that indicates the proliferation/differentiation states in human MSCs. Moreover, differences in the relative NS protein and mRNA levels may reflect the degree of proliferation and can be used to characterize in vitro expansion capabilities.