Bacillus subtilis LmrA and QdoR (formerly YxaF) are paralogous transcriptional regulators that repress their regulon comprising the lmrAB operon, the qdoR gene, and the qdoI-yxaH operon, by binding to the LmrA/QdoR boxes located in the promoter regions. Detachment of them followed by derepression of the target genes is induced by certain flavonoids. To identify the residues critical to the ligand response in QdoR, we selected eight residues based on structural information, produced eight single-mutated QdoRs in which each residue was replaced with alanine, and evaluated their capacities for DNA binding and the flavonoid response by gel retardation analysis. The three mutants, carrying the alanine substitution at Phe87, Trp131, or Phe135, showed features distinctly different from those of the wild type and from each other. We further examined the in vivo function of the mutant with alanine substitution at Trp131 by reporter assay. This largely supported the corresponding in vitro results. The in vitro and in vivo data suggest that Phe87, Trp131, and Phe135, forming a hydrophobic cluster in QdoR, play crucial roles in the DNA binding, flavonoid accommodation, and/or conformational change triggered by ligand binding.