Recent studies have demonstrated that natural water that has 65% of the deuterium concentration depleted, can exhibit anti-tumor properties. However, the anti-tumor effects of DDW on various nasopharyngeal carcinoma (NPC) cells have not previously been reported. In the present study, NPC cell lines and normal preosteoblast MC3T3-E1 cells were grown in RPMI1640 media containing different deuterium concentrations (50–150ppm). The effects of DDW on the proliferation and migration of NPC and MC3T3-E1 cells were investigated using the MTT, plate colony formation, and Transwell assays, as well as Boyden chamber arrays, flow cytometry (FCM), western blot and immunofluorescence. We found that DDW was an effective inhibitor of NPC cell proliferation, plated colony formation, migration and invasion. In contrast, the growth of normal preosteoblast MC3T3-E1 cells was promoted when they were cultured in the presence of DDW. Cell cycle analysis revealed that DDW caused cell cycle arrest in the G1/S transition, reduced the number of cells in the S phase and significantly increased the population of cells in the G1 phase in NPC cells. Western blot analysis revealed that treatment with DDW significantly increased the expression of NADPH:quinone oxidoreductase-1 (NQO1), while immunofluorescence assay analysis revealed that treatment with DDW decreased the expression of PCNA and matrix metalloproteinase 9 (MMP9) in NPC cells. These results demonstrated that DDW is a novel, non-toxic adjuvant therapeutic agent that suppresses NPC cell proliferation, migration, and invasion by inducing the expression of NQO1 and causing cell cycle arrest, as well as decreasing PCNA and MMP9 expression.