ABSTRACTChemotaxis is an important virulence factor in some enteric pathogens, and it is involved in the pathogenesis and colonization of the host. However, there is limited knowledge regarding the environmental signals that promote chemotactic behavior and the sensing of these signals by chemoreceptors. To date, there is no information on the ligand molecule that directly binds to and is sensed byCampylobacter jejuniTlp1, which is a chemoreceptor with a dCache-type ligand-binding domain (LBD). dCache (doubleCalcium channels andchemotaxis receptor) is the largest group of sensory domains in bacteria, but the dCache-type chemoreceptor that directly binds to formate has not yet been discovered. In this study, formate was identified as a direct-binding ligand ofC. jejuniTlp1 with high sensing specificity. We used the strategy of constructing a functional hybrid receptor ofC. jejuniTlp1 and theEscherichia colichemoreceptor Tar to screen for the potential ligand of Tlp1, with the binding of formate to Tlp1-LBD being verified using isothermal titration calorimetry. Molecular docking and experimental analyses indicated that formate binds to the membrane-proximal pocket of the dCache subdomain. Chemotaxis assays demonstrated that formate elicits robust attractant responses of theC. jejunistrain NCTC 11168, specifically via Tlp1. The chemoattraction effect of formate via Tlp1 promoted the growth ofC. jejuni, especially when competing with Tlp1- or CheY-knockout strains. Our study reveals the molecular mechanisms by whichC. jejunimediates chemotaxis toward formate, and, to our knowledge, is the first report on the high-specificity binding of the dCache-type chemoreceptor to formate as well as the physiological role of chemotaxis toward formate.