Objective(s): The mesenchymal stem cells (MSCs) have been introduced as appropriate cells for tissue engineering and medical applications. Some studies have shown that topography of materials especially physical surface characteristics and particles size could enhance adhesion and proliferation of osteoblasts. In the present research, we studied the distinction effect of 30 and 60 μg/ml of zinc oxide (ZnO) on differentiation of human mesenchymal stem cells to osteoblast. Materials and Methods: After the third passage, human bone marrow mesenchymal stem cells were exposed to 30 and 60 μg/ml of ZnO nanoparticles having a size of 30 nm. The control group has received no ZnO nanoparticles. On day 15 of incubation for monitoring the cellular differentiation, alizarin red staining and RT-PCR assays were performed to evaluate the level of osteopontin, osteocalsin and alkaline phosphatase genes expression. Results:In the group receiving 30 μg/ml of ZnO nanoparticles, the expression of osteogenic markers such as alkaline phosphatase, osteocalcin and osteopontin genes were significantly higher than both control and the group receiving 60 μg/ml ZnO nanoparticle. These data also confirmed by alizarin red staining. Conclusion: It seems the process of differentiation of MSCs affected by ZnO nanoparticles is dependent on dose as well as on the size of ZnO.