Pyridoxal 5′‐phosphate (PLP), the well‐known active form of vitamin B₆, is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N‐terminal helix‐turn‐helix motif that binds DNA and an effector‐binding C‐terminal domain homologous to PLP‐dependent enzymes. Although it is known that PLP acts as an anti‐activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA‐binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B₆ vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA‐binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.