Acetic acid bacteria grow while producing acetic acid, resulting in acidification of the culture. Limited reports elucidate the effect of changes in intracellular pH on transcriptional factors. In the present study, the intracellular pH of Komagataeibacter europaeus was monitored with a pH-sensitive green fluorescent protein, showing that the intracellular pH decreased from 6.3 to 4.7, accompanied by acetic acid production during cell growth. The leucine-responsive regulatory protein of K. europaeus ( Ke Lrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog ( Ec Lrp) at different pH levels. The DNA-binding activities of Ec Lrp and Ke Lrp with the target DNA ( Ec-ilvI and Ke-ilvI ) were examined by gel mobility shift assays under various pH conditions. Ec Lrp showed the highest affinity with the target at pH 8.0 ( K d , 0.7 μM), decreasing to a minimum of 3.4 μM at pH 4.0. Conversely, Ke Lrp did not show significant differences in binding affinity between pH 4 and 7 ( K d , 1.0–1.5 μM), and the highest affinity was at pH 5.0 ( K d , 1.0 μM). Circular dichroism spectroscopy revealed that the α-helical content of Ke Lrp was the highest at pH 5.0 (49%), and was almost unchanged while maintaining >45% over a range of pH levels examined, while that of Ec Lrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that Ke Lrp is stable and functions over a wide range of intracellular pH levels. IMPORTANCE Lrp is a highly conserved transcriptional regulator found in bacteria and archaea and regulates transcriptions of various genes. The intracellular pH of acetic acid bacteria (AAB) changes accompanied by acetic acid production during cell growth. The Lrp of AAB K. europaeus ( Ke Lrp) was structurally stable over a wide range of pH and maintained DNA-binding activity even at low pH compared with Lrp from E. coli living in a neutral environment . In vitro experiment showed DNA-binding activity of Ke Lrp to the target varied with changes in pH. In AAB, change of the intracellular pH during a cell growth would be an important trigger in controlling the activity of Lrp in vivo .