Abstract. Objectives: Nucleostemin (NS) is a recently identified GTP‐binding protein, predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS is expressed in bone marrow‐derived mesenchymal stem cells, and its expression ceases upon induction of neural differentiation. The major aim of this study was to determine whether down‐regulation of NS expression acts as a promoter, or otherwise as a by‐product of differentiation and senescence processes. Materials and methods: We used RNA interference protocols to specifically knock down NS in rat bone marrow‐derived stromal stem cells. Changes in rate of proliferation and cell cycle profile after knocking‐down of NS were measured. In addition, changes in expression of associated genes were studied by semiquantitative RT‐PCR, Western blotting and immunocytochemistery. Results: Knocked‐down expression of NS caused a significant decrease in the rate of cell proliferation with concomitant shutting off of expression of cyclin D1 and survivin, two other well‐known regulators of cell proliferation. Interestingly, we noticed no obvious changes in expression level of p21, the main effector of p53 for its cell cycle repressing function. Conclusion: Our findings revealed a master role for NS in promoting proliferation of rat bone marrow‐derived stromal stem cells. Moreover, we suggest that despite previous proposals, the cell cycle arrest/inhibitory role of NS is unlikely to be related to its proposed property of interaction with p53.