The purpose of this study was to investigate the influence of nitro oxide (NO) from mesenchymal stem cells (MSC) on the proliferative responses of allogeneic lymphocytes and its mechanism. MSCs were isolated and cultured from human bone marrow. Selected surface antigens of MSCs were detected by flow cytometry and their morphologic characteristics were determined by microscopy. Mitomycin C-treated MSCs were plated in dishes and then mixed lymphocyte cultures (MLC) were set up. After 4 days, lymphocyte proliferation was determined by CCK-8 assays; NO secretion in coculture supernatant was determined by Griess reagent kit; the level of FOXP3 mRNA expression was detected by real-time quantitative PCR. The results indicated that in MSC/MLC coculture experiment, the lymphocyte proliferation decreased significantly with of IOD value 0.49+/-0.03, NO production increased obviously (21.05+/-1.14 micromol/L) and FOXP3 mRNA expression was increased [(1.56+/-0.34)%] as compared with MLC coculture without MSC. There were significant difference between these two groups. It is concluded that NO production in human MSC culture up-regulates FOXP3 mRNA expression and thus inhibits lymphocyte proliferation response.