<jats:p>Pseudouridine is a noncanonical C–nucleoside commonly present in <jats:styled-content style="fixed-case">RNA</jats:styled-content>, which is not metabolized in mammals, but can be recycled by the unique enzyme family of bacterial pseudouridine glycosidases such as YeiN from <jats:italic>Escherichia coli</jats:italic>. Here, we present rigorous bioinformatic and biochemical analyses of the protein family in order to find sequences that might code for nonpseudouridine glycosidase activities. To date, the only other function reported for the enzyme family occurs during the biosynthesis of the antibiotic alnumycin A in <jats:italic>Streptomyces</jats:italic> species, where AlnA functions as an unusual C–glycosynthase. Bioinformatics analysis of 755 protein sequences identified one group of sequences that were unlikely to harbour pseudouridine glycosidase activities. This observation was confirmed <jats:italic>in vitro</jats:italic> with one representative protein, IdgA from <jats:italic>Streptomyces albus</jats:italic>, which was unable to synthesize pseudouridine monophosphate, but was able to attach <jats:sc>d</jats:sc>–ribose‐5‐phosphate to juglone. Furthermore, our analyses provide evidence for horizontal gene transfer of pseudouridine glycosidases that may have occurred in <jats:italic>Streptomyces</jats:italic> and <jats:italic>Doria</jats:italic> species. Inspection of the genomic loci in the vicinity of pseudouridine glycosidases revealed that in 77% of the strains a kinase gene putatively involved in the phosphorylation of pseudouridine was found nearby, whereas the sequences encoding nonpseudouridine glycosidases coexisted with a phosphatase of the haloacid dehalogenase enzyme family. The investigation suggested that these unknown sequences might be involved in the biosynthesis of soluble blue pigments because of the presence of genes homologous to nonribosomal peptide synthetases.