The HxlR protein from Bacillus subtilis belongs to the DUF24 protein family (InterPro No. IPR002577) of unknown function. The hxlR gene that encodes this protein is located upstream of the hxlAB operon. This operon encodes two key enzymes in the ribulose monophosphate pathway that are involved in formaldehyde fixation, 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Expression of the hxlAB operon is induced by the presence of formaldehyde. Recombinant HxlR prepared from Escherichia coli showed specific binding to a region of DNA upstream of the hxlAB operon. Using gel-retardation and DNase I footprinting assays, we identified two 25 bp binding regions for HxlR within the upstream DNA. Surface plasmon resonance analyses suggested that two HxlR dimers sequentially bound to the DNA. Finally, we demonstrated that each of the two binding regions for HxlR was necessary for formaldehyde-induced expression of the hxlAB operon in B. subtilis. Thus, we have shown that HxlR is a DNA-binding protein that is necessary for formaldehyde-induced expression of hxlAB in B. subtilis.