To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we sought the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (O₂) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (m) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this 2-dimensional tissue model, m was abolished in cells located approximately >500 µm from the oxygen source (anoxic front, AF), indicating limitations in diffusional oxygen delivery. This result perfectly matched O₂ determined by GFP. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain containing protein (PHD) inhibitor, AF was expanded to 1500-2000 µm from the source. In these cells, tissue O₂ were substantially decreased, indicating that the PHD pathway activation suppressed mitochondrial respiration. The expansion of AF and the reduction of O₂ were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain m despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding AF, although the tissue O₂ remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining _m in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain _m in an otherwise anoxic microenvironment by decreasing tissue O₂ while activating oxygen-independent electron transport in mitochondria.