Recent studies have shown that the depletion of naturally occurring deuterium can result in tumor regression. The aim of the present study was to show the growth inhibitory effects of DDW discretely and in combination with 5-FU on MCF-7 breast cancer cells and to determine possible mechanisms underlying these changes. MCF7 cells were grown in RPMI medium with decreasing deuterium concentrations of DDW individually, 5-FU alone and both for 24, 48, and 72 h. Cell viability was determined with the MTT assay. The cell cycle and antioxidant enzymes status were measured using flow cytometry and quantitative luminescence methods, respectively. Our results showed that treatment with DDW especially in 30-100 ppm concentrations imposed the highest cell growth inhibitory effect. The cell cycle analysis revealed that DDW caused the cell cycle arrest in the G1/S transition, reduced the number of the cells in the S phase and significantly increased the population of cells in the G1 phase in MCF-7 cells. The activities of superoxide dismutase (SOD) and catalase (CAT) enzymes also increased in the same low concentrations of DDW. In conclusion, DDW can open new strategic approach in breast cancer therapy. Highlights DDW cause lethality in cancer cells. DDW augmented 5-FU inhibitory effects on breast cancer cell lines. Cell inhibitory results lead to the discovery of synergic effects of DDW-drug combinations Synergistic anti cancer effects of DDW with 5-FU is enhanced by decreasing deuterium content of the DDW. DDW exerts effects on the cell cycle, changes in cell configuration and induces antioxidant enzymes in vitro. DDW can be considered as an adjuvant to conventional anticancer agents in future trials.