Comamonas testosteroni CNB-1 behaves chemotactically toward a wide range of organic compounds, and 19 methyl-accepting chemotaxis proteins (MCPs) were annotated from the genome of strain CNB-2, a plasmid-curing derivative from strain CNB-1. The MCP-free mutant CNB-1Δ20 completely lost its chemotactic responses. In this study, we found that a chemoreceptor, namely MCP2983, restored chemotactic responses toward nine carboxylic acids and ten aromatic compounds to CNB-1Δ20. Isothermal titration calorimetry analysis indicated that the ligand-binding domain (LBD) of MCP2983 specifically binds to cis-aconitate but not other tested compounds. Deletion of the LBD of MCP2983 impaired chemotactic responses toward cis-aconitate as well as other tested compounds, indicating that the LBD of MCP2983 was essential for triggering chemotactic responses. Five amino acid residues (M(81), S(156), E(157), I(158), and L(159)) that are located at a putative ligand-binding pocket were identified to be involved in binding to cis-aconitate. So far, the MCP2983 represents the sole biochemically identified chemoreceptor that specifically binds to cis-aconitate and is able to trigger chemotaxis towards diverse organic compounds.