Cobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all fungal lineages, except for Dikarya. This observation is supported by the genomic presence of both B12 dependent enzymes and cobalamin modifying enzymes. Moreover, the genes identified are actively transcribed in many taxa. Most fungal cobalamin dependent enzymes and cobalamin metabolism proteins are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin trait with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and likely in the LECA. ### Competing Interest Statement The authors have declared no competing interest.

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