With more than 600,000 proteins included in its current release, the radical S-adenosyl-l-methionine (SAM) superfamily constitutes arguably the largest superfamily of enzymes [1]. These metalloenzymes, recognized twenty years ago as a unified superfamily [4], have been shown to catalyze an impressive diversity of radical-based reactions in a myriad of biosynthetic pathways including vitamin, cofactor and metallic center biosynthesis [5, 6, 7, 8, 9, 10], nucleic acid modifications, DNA repair [11], protein post-translational modifications [12, 13, 14] along with a central role in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPP) [2]. As typical features, radical SAM enzymes share a partial (β/α)6 or a full (β/α)8 triose phosphate isomerase (TIM) barrel fold, a [4Fe–4S] cluster and the dependence to the S-adenosyl-l-methionine cofactor [2,15]. However, a major discovery of the last decade is that in addition to the radical SAM domain, most of these enzymes possess additional domains housing metallic centers such as iron-sulfur clusters [16,17] and cobalamin (vitamin B12) [18, 19, 20], expanding their chemical repertoire. For instance, more than 200,000 distinct radical SAM enzymes are predicted to be dependent on the cobalamin cofactor. These B12-dependent radical SAM enzymes, which now delineate a superfamily on their own [18,21, 22, 23, 24, 25, 26, 27, 28, 29∗, 30∗∗], are involved in major biosynthetic pathways including protein post-translational modification [30∗∗, 31, 32], antibiotic (fosfomycin [33], carbapenem [22], gentamicin [34] and oxetanocin A [26]) and RiPP [18,23,35] biosynthesis. However, after more than a decade of investigation, it is only recently that the first structures of B12-dependent radical SAM enzymes in complex with their substrates have been solved, revealing unexpected catalytic and structural features. This review highlights recent advances in our understanding of these unique biocatalysts from a biochemical and structural perspective.