Objective: Nucleostemin (NS) is a recently identified gene that is expressed mainly in the nucleoli of neuronal, embryonic and mesenchymal stem cells which plays a role in their self renewal. Over expression of this gene has been reported in several cancer cell lines tumoral tissues including gastric, hepatic and prostate cancer. In this study, we investigated the effects of suppression of this gene by RNAi technique in a human bladder cancer cell line. Materials and Methods: After confirmation of expression of nucleostemin in the bladder cancer cell line 5637, 21-mer oligoes against NS were transfected into cells and suppression of NS was confirmed by RT-PCR, immunocytochemistry (ICC) and western blotting. Proliferation assays were done by counting the cell numbers. Changes in cell cycle and apoptosis induction were assessed by staining cells with Propidium Iodide and Annexin V-FITC respectively. Results: Our results revealed that nucleostemin is highly expressed in 5637 carcinoma cells. Expression of NS, by semi-quantitative RT-PCR, after treatment with specific oligoes against it, decreased around 85% in comparison to control groups. In addition, ICC and western blotting further confirmed the suppression of nucleostemin at the protein levels. Analysis of cell proliferation assays showed a considerable decline in the number of cells, specially, 72 h after treatment of cells with oligoes against NS. Cell cycle analysis after NS suppression showed increase of sub-diploid events, characteristic of apoptotic cells that were confirmed by staining cells with Annexin V-FITC dye. Conclusion: It seems that nucleostemin expression plays an important role in the induction of cell proliferation as well as inhibition of apoptosis occurrence in 5637 cells. Further studies focusing on suppression of this gene in vivo, may help to find potential therapeutic strategies to cure bladder cancer.