LldR is a lactate-responsive transcription factor (TF) that transcriptionally regulates thelldPRDoperon consisting of lactate permease and lactate dehydrogenase. ThelldPRDoperon facilitates the utilisation of lactic acid in bacteria. However, the role of LldR in whole genomic transcriptional regulation, and the mechanism involved in adaptation to lactate remains unclear. We used genomic SELEX (gSELEX) to comprehensively analyse the genomic regulatory network of LldR to understand the overall regulatory mechanism of lactic acid adaptation of the model intestinal bacteriumEscherichia coli. In addition to the involvement of thelldPRDoperon in utilising lactate as a carbon source, genes related to glutamate-dependent acid resistance and altering the composition of membrane lipids were identified as novel targets of LldR. A series ofin vitroandin vivoregulatory analyses led to the identification of LldR as an activator of these genes. Furthermore, the results of lactic acid tolerance tests and co-culture experiments with lactic acid bacteria suggested that LldR plays a significant role in adapting to the acid stress induced by lactic acid. Therefore, we propose that LldR is anl-/d-lactate sensing TF for utilising lactate as a carbon source and for resistance to lactate-induced acid stress in intestinal bacteria.