Cyclophilin D (CypD) is a mitochondrial matrix protein implicated in cell death, but a potential role in bioenergetics is not understood. Here, we show that loss or depletion of CypD in cell lines and mice induces defects in mitochondrial bioenergetics due to impaired fatty acid β-oxidation. In turn, CypD loss triggers a global compensatory shift towards glycolysis, with transcriptional upregulation of effectors of glucose metabolism, increased glucose consumption and higher ATP production. In vivo, the glycolytic shift secondary to CypD deletion is associated with expansion of insulin-producing β-cells, mild hyperinsulinemia, improved glucose tolerance, and resistance to high fat diet-induced liver damage and weight gain. Therefore, CypD is a novel regulator of mitochondrial bioenergetics, and unexpectedly controls glucose homeostasis, in vivo.