CLYBL encodes a ubiquitously expressed mitochondrial enzyme， conserved across all vertebrates， whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals， with reduced circulating B levels being the only and consistent phenotype reported to date. Here， by combining enzymology， structural biology， and activity-based metabolomics， we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA， an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate， a recently identified human anti-microbial metabolite and immunomodulator. We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B metabolism and that itaconyl-CoA is a cofactor-inactivating， substrate-analog inhibitor of the mitochondrial B-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B inactivation.