Methotrexate (MTX) is known antagonist of folic acid and widely used as an anti-cancer drug. The folate receptor (FR) and reduced folate carrier are mostly responsible for internalization of methotrexate in tumor cells. Mutation in reduced folate carrier (RFC) leads to resistance against MTX in various tumor cell lines including MDA-MB-231 breast cancer cells. To overcome the resistance of MTX, folate receptor targeted nanoparticles have been commonly used for targeting breast tumors. The aim of the study is to determine the ability of methotrexate gold nanoparticles (MTX-GNPs) in the induction of apoptosis and to explore the molecular changes at genomics and proteomics level. Different assays like cell viability assay, cell cycle analysis, apoptosis, real-time PCR and western blot were carried out to evaluate the anti-cancer effect of MTX-Gold NPs on MCF-7 and MDA-MB-231 cells. Our observations demonstrated the decrease in the percent viable cells after the treatment of MTX-GNPs, with an arrest in cell cycle at G0/G1 phase and a significant increase in apoptotic cell population and loss of mitochondrial membrane potential in MCF-7 and MDA-MB-231 cells. Folate receptor targeted MTX-GNPs showed significant cellular uptake in breast cancer cells along with significant down-regulation in expression of anti-apoptotic gene (Bcl-2) and up-regulation in expression of pro-apoptotic genes (Bax, Caspase-3, Caspase-9, APAF-1, p53). These results unveil the increased anti-cancer effect of MTX-GNPs in cancer cells.