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Deuterium, a stable isotope of hydrogen present in natural water at ~150 ppm, has been implicated in modulating cellular metabolism and tumor progression. While deuterium-depleted water (DDW) has shown anticancer effects in preclinical and clinical studies, the underlying transcriptional mechanisms remain incompletely defined. Here, we profiled gene expression in A549 lung adenocarcinoma cells cultured for 72 hours in media containing four graded deuterium concentrations (40, 80, 150, and 300 ppm) using a targeted NanoString panel of 236 cancer-related genes. After stringent quality filtering, 87 genes were retained and classified into nine distinct expression patterns based on fold-change trends relative to the 150 ppm control. High deuterium (300 ppm) induced strong upregulation of oncogenic and survival-related genes (e.g., EGFR, CTNNB1, STAT3, CD44), while DDW (40–80 ppm) led to selective downregulation of oncogenes (e.g., MYCN, ETS2, IRF1) and drug-resistance genes (e.g., ABCB1). Several genes involved in DNA repair, apoptosis, and extracellular matrix remodeling exhibited dose-dependent responses, suggesting coordinated regulation by deuterium abundance. These findings demonstrate that deuterium concentration functions as a biologically active variable capable of modulating cancer-relevant gene networks. This exploratory dataset refines mechanistic models of DDW action and provides a foundation for future studies incorporating biological replication, functional assays, and in vivo validation. Significance: Deuterium concentration modulation alters oncogenic, apoptotic, and drug-resistance gene networks in lung adenocarcinoma cells, refining prior models of deuterium-depleted water effects. These findings identify deuterium concentration as a biologically active variable warranting further mechanistic and translational investigation.