Glyoxylate is a key metabolite generated from various precursor substrates in different subcellular compartments including mitochondria, peroxisomes, and the cytosol. The fact that glyoxylate is a good substrate for the ubiquitously expressed enzyme lactate dehydrogenase (LDH) requires the presence of efficient glyoxylate detoxification systems to avoid the formation of oxalate. Furthermore, this detoxification needs to be compartment-specific since LDH is actively present in multiple subcellular compartments including peroxisomes, mitochondria, and the cytosol. Whereas the identity of these protection systems has been established for both peroxisomes and the cytosol as concluded from the deficiency of alanine glyoxylate aminotransferase (AGT) in primary hyperoxaluria type 1 (PH1) and glyoxylate reductase (GR) in PH2, the glyoxylate protection system in mitochondria has remained less well defined. In this manuscript, we show that the enzyme glyoxylate reductase has a bimodal distribution in human embryonic kidney (HEK293), hepatocellular carcinoma (HepG2), and cervical carcinoma (HeLa) cells and more importantly, in human liver, and is actively present in both the mitochondrial and cytosolic compartments. We conclude that the metabolism of glyoxylate in humans requires the complicated interaction between different subcellular compartments within the cell and discuss the implications for the different primary hyperoxalurias.