AbstractThe pyridoxal 5′‐phosphate binding protein (PLP‐BP) is believed to play a crucial role in PLP homeostasis, which may explain why it is found in living organisms from all kingdoms. Escherichia coli YggS is the most studied homolog, but human PLP‐BP has also attracted much attention because variants of this protein are responsible for a severe form of B6‐responsive neonatal epilepsy. Yet, how PLP‐BP is involved in PLP homeostasis, and thus what its actual function is in cellular metabolism, is entirely unknown. The present study shows that YggS binds RNA and that the strength of this interaction is modulated by PLP. A key role in RNA binding is clearly played by Lys137, an invariant residue located on a protein loop away from the PLP binding site, whose importance has been highlighted previously. The interaction with RNA is evidently conserved, since it is also observed with human PLP‐BP. The RNA binding site, which is apparently located at the entrance of the PLP‐binding site, is also evolutionarily conserved. It is therefore reasonable to assume that PLP, by defining the conformation of the protein, determines the RNA binding affinity. RNA‐seq analysis of RNA co‐purified with or captured by YggS revealed SsrA and RnpB RNAs, respectively involved in trans‐translation and tRNA maturation, as the major molecular components. This work opens up new horizons for the function of the PLP‐BP, which could be related to its interaction with RNA and modulated by PLP, and thus play a role in an as yet unknown regulatory mechanism.