AbstractThe micronutrient vitamin B12 is an essential cofactor for two enzymes: methionine synthase, which plays a key role in the one-carbon cycle; and methylmalonyl-CoA mutase, an enzyme in a pathway that breaks down branched-chain amino acids and odd-chain fatty acids. A second, vitamin B12-independent pathway that degrades propionic acid was recently described inCaenorhabditis elegans, the propionate shunt pathway. Activation of five shunt pathway genes in response to low vitamin B12 availability or high propionic acid levels is accomplished by a transcriptional regulatory mechanism involving two nuclear hormone receptors, NHR-10 and NHR-68. Here, we report that theC. elegansMediator subunitmdt-15is also essential for the activation of the propionate shunt pathway genes, likely by acting as a transcriptional coregulator for NHR-10.C. elegans mdt-15mutants fed with a low vitamin B12 diet have transcriptomes resembling those of wild-type worms fed with a high vitamin B12 diet, with low expression of the shunt genes. Phenotypically, the embryonic lethality ofmdt-15mutants is specifically rescued by diets high in vitamin B12, but not by dietary polyunsaturated fatty acids, which rescue many other phenotypes of themdt-15mutants. Finally, NHR-10 binds to MDT-15 in yeast two-hybrid assays, and the transcriptomes ofnhr-10mutants share overlap with those ofmdt-15mutants. Our data show that MDT-15 is a key coregulator for an NHR regulating propionic acid detoxification, adding to roles played by NHR:MDT-15 partnerships in metabolic regulation and pinpointing vitamin B12 availability as a requirement formdt-15dependent embryonic development.