Objective: Nano-formulation approaches would be more efficient in decreasing drug toxicity, preventing tumor development in relation to free form of drug.Methods: Therefore, the purpose of this study is to construct poly lactic-coglycolic acid (PLGA) nanoparticles (NPs) loaded with Metformin (MET) to investigate their cytotoxicity as well as their impact on expression levels of apoptosis related genes in MDA-MB-231 breast cancer cells. Field Emission Scanning Electron Microscope (FE-SEM) and Fourier-transform infrared spectroscopy (FTIR) characterized the synthesized NPs. Then, MTT assay was used to evaluate and compare the cytotoxic effect of various concentrations of the chemotherapeutic molecules in pure and nano-formulated forms after 48 h exposure time. Moreover, the mRNA levels of apoptosis related genes expression were studied by quantitative PCR.Results: By encapsulating MET into PLGA, the cytotoxic efficiency of the compounds considerably augmented for all concentrations. Furthermore, the results demonstrated that MET-loaded NPs could induce apoptosis in MDA-MB-231 breast cancer cell by upregulation of caspase-3, caspase-7 and BAX, along with Bcl-XL, hTERT and Cyclin D1 down regulation.