The gram-negative bacterium Lonsdalea populi causes an emerging poplar (Populus × euramericana) canker resulting in severe losses to poplar production in China and Europe. Two-component signal transduction systems play important roles in the regulation of virulence and stress responses in phytopathogenic bacteria. We identified a two-component pair (Lqp2625-Lqp2624) in L. populi, highly homologous to DcuS-DcuR of Escherichia coli. Mutants lacking DcuS or DcuR displayed normal growth while their virulence on poplar twigs was impaired. An inability to produce flagella indicated that DcuS and DcuR are involved in biofilm formation and swimming motility. Moreover, the loss of DcuS or DcuR led to increased sensitivity to oxidative stress and chloramphenicol through downregulation of genes associated with catalases and the multidrug efflux pump, suggesting that the two-component pair contributes to cellular adaptation to oxidative and antibiotic stresses. We identified key domains and putative phosphorylation sites important for virulence and stress responses. Our findings reveal the functions of DcuS-DcuR in virulence and stress responses in L. populi and provide increasing evidence that two-component systems are crucial during the infection process and stress adaptation in bacteria.