Many disparate studies have reported the ambiguous role of hydrogen sulfide (H2 S) in cell survival. The present study investigated the effect of H2 S on the viability of cancer and non-cancer cells. Cancer and non-cancer cells were exposed to H2 S [using sodium hydrosulfide (NaHS) and GYY4137] and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi ) was determined by ratiometric pHi measurement using BCECF staining. Continuous, but not a single, exposure to H2 S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H2 S-releasing donor, GYY4137, significantly increased glycolysis, leading to overproduction of lactate. H2 S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification, leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells. Low and continuous exposure to H2 S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.