Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Since acidity contributes to the acquisition of various malignant phenotypes by cancer cells, therapeutic strategies that target cancer cells might be more effective in an acidic microenvironment. We report here that drugs which abrogate mitochondrial respiration have enhanced cytotoxicity in an acidic extracellular pH in normoxia, independent of P53 or BRAF (V600E) mutations and/or resistance against BRAF inhibition with vemurafenib. In contrast, the same mitochondrial inhibitors induced less cytotoxicity in the same cells at a neutral or alkaline extracellular pH. Surprisingly, cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from recently reported mediators, including PGC1alpha-driven mitochondrial addiction, therapy-induced senescence and JARID1B-high slow-cycling status, which have been shown to promote vulnerability against mitochondrial inhibition. Cleavage of caspase-3 and caspase-6 were significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH. Furthermore, intracellular Ca2+ level was increased and an intracellular Ca2+ chelator BAPTA/AM partially suppressed cytoplasmic Ca2+ level as well as cell death. Interestingly, ROS scavengers synergized to promote apoptosis after mitochondrial inhibition at an acidic pH, suggesting that ROS mediate cell survival in this context. Finally and importantly, systemic alkali buffering with NaHCO3 rescued both subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. These data suggest that extracellular pH acts as a threshold that modulates the cytotoxicities of mitochondrial inhibitors against melanoma.