Objective: Nitric oxide (NO) is a free radical and a signaling molecule which controls many cellular and physiological mechanisms such as; blood vessel contraction, blood pressure and immunological response. In previous studies it was shown that the short time treatment of mesenchymal stem cells (MSCs) with sodium nitroprusside (SNP) as an NO releasing agent at low concentration did not affect the cell viability, but caused metabolic imbalance. The aim of this study was to investigate the effect of low concentration of SNP for long time on cell viability, proliferation, cell cycle and expression of the genes involved in cell cycle. Materials and Methods: MSCs after the third passage was treated every 1 hour in each 72 hours with 100 μM of SNP. After 5, 10, 15 and 20 days, the viability and proliferation of the cells was estimated using MTT, Tripan blue and PDN, CFA method. In addition, we used flow cytometry to study the cell cycle whereas the profile of the proteins was evaluated using electrophoresis. The expression of Raf1, Cdk2, Cdk4, P53 and GAPDH genes involves in the cell cycle was determined using quantitative real-time PCR. Also, we checked the effect of the repeated passages on the control and treated of MSCs growth. Results: Cell treated with SNP caused the reduction of viability at 5, 10, 15 and 20 days. The same treatment caused the number of the colonies to reduce significantly at all treatment times, but no changes were observed in diameter of the colonies. In addition, the cell cycle was 93% arrested at G1 stage following SNP treatment at day 20. In the MSCs treated with SNP, the expression of the Cdk2 and Cdk4 was reduced; whereas the expression of P53 was elevated and the Raf1 expression remained the same. In the treated cells we observed changes in the density of some polypeptides, where two polypeptides with the molecular weight of 16.73 and 13.40 were expressed only in the treated group, the cell treated with SNP tolerated up to 11 passages and
