2-deoxy-D-Glucose (2DG) causes cytotoxicity in cancer cells by disrupting thiol metabolism. It is an effectivecomponent in therapeutic strategies. It targets the metabolism of cancer cells with glycolysis inhibitory activity.On the other hand, MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8activating enzyme), inactivates SCF E3 ligase and causes accumulation of its substrates which triggers apoptosis.Combination of these components might provide a more efficient approach to treatment. In this research,2DG and MLN4924 were co-applied to breast cancer cells (MCF-7 and SKBR-3) and cytotoxic and apoptoticactivity were evaluated the by Micro culture tetrazolium test (MTT), TUNEL and ELISA methods. Caspase3and Bcl2 genes expression were evaluated by real time Q-PCR methods. The results showed that MLN4924 andMLN4924/2DG dose-dependently suppressed the proliferation of MCF7 and SKBR-3 cells. Cell survival of breastcancer cells exposed to the combination of 2DG/MLN4924 was decreased significantly compared to controls(p<0.05), while 2DG and MLN4924 alone had less pronounced effects on the cells. The obtained results suggestthat 2DG/MLN4924 is much more efficient in breast cancer cell lines with enhanced cytotoxicity via inducing aapoptosis cell signaling gene, caspase-3.