The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata. Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the Delta cheY1 mutant exhibited decreased frequency of direction reversal, whereas the Delta cheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the Delta cheY1 and Delta cheY2 mutants was lower than that in the WT, but the Delta cheY2 mutant had more obvious EPS defects that were similar to those of the Delta cheY1 Delta cheY2 and Delta eps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of Delta cheY1 and Delta cheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the Delta cheY2 mutant was severely impaired compared to that of the Delta cheY1 mutant. Taken together, the Delta cheY2 phenotypes are more severe than the Delta cheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the Delta cheY2 single-mutant phenotype. These defects of Delta cheY1 and Delta cheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions. IMPORTANCE Azorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.