BACKGROUND Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. METHODS We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. RESULTS Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. CONCLUSIONS These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration. CONCLUSIONS Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches.