Alterations in cellular metabolism are among the most consistent hallmarks of cancer. Herein we have investigated the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, lactate dehydrogenase A (LDH-A) levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.