Cancer persists as one of the primary global causes of death, highlighting the pressing demand for innovative therapeutic methods. Deuterium-depleted water (DDW) has emerged as a potential anticancer agent due to its ability to influence cellular processes. This study explores the inhibitory impacts of DDW with diverse concentrations on the proliferation of lung (A549) and liver (HepG2) cancer cells and the migration of breast (MCF-7) cancer cells. It also examines its apoptosis effects on A549 and MCF-7 cancer cells. Using the CCK-8 assay, real-time cell analysis, fluorescence microscopy, and wound-healing assays, the impact of DDW on tumor cells was systematically evaluated. The CCK-8 assay demonstrated that DDW significantly inhibited tumor cell growth, a finding corroborated by real-time cell growth curves. Fluorescence microscopy revealed that DDW triggered apoptosis and necrosis in A549 and MCF-7 cells. In addition, the wound - healing assays demonstrated that, compared with the control group, DDW at concentrations of 0 ppm, 30 ppm, 60 ppm, and 100 ppm significantly weakened the migration ability of MCF-7 cells, and all these differences were statistically significant (P < 0.05). These results suggest that DDW effectively suppresses tumor cell proliferation and migration while promoting apoptosis. This study suggests that DDW may have potential to inhibit tumor cell proliferation as an adjuvant therapy, and it provides a solid basis for subsequent research on the mechanisms and possible therapeutic applications of DDW in cancer care.