OBJECTIVE: Green Fluorescent Protein (GFP) has been used as a marker of gene expression and a single cell marker in living organisms in cell biology studies. The important areas that GFP is used are expression levels of different genes in different organisms by inserting GFP in these genes and as a marker in living cells. In this study, we tried to optimize transfection of mesenchymal stem cells, (MSCs) used for regeneration of damaged tissues in animals, by GFP containing plasmid vector by which MSCs can be followed in vivo.METHODS: To this aim, phM-GFP plasmid vector carrying GFP gene and effectene transfection reagent were used. RESULTS: The data revealed that twice transfection of MSCs resulted in higher expression of GFP for longer times as compared to once transfected MSCs. On the other hand, leaving the chemical transfection agents in the medium induced apoptosis after a while. CONCLUSION: As a conclusion we suggest the transfection of MSCs twice with 48 hours interval and removal of transfection agents after 8 hours which removed toxic and apoptotic effects of the chemicals.