Burkholderia pseudomallei is the cause of melioidosis, a serious invasive disease of humans and other animal species with a mortality rate of 10-50%. Screening of a B. pseudomallei transposon library identified a transposon mutant that was significantly attenuated for virulence in BALB/c mice. This mutant had a transposon insertion in the gene bvrS (BPSS0687), predicted to encode the sensor kinase component of a two-component signal transduction systems (TCSTS). TCSTS are important regulators of bacterial gene expression. An independent, directed bvrS mutant was also highly attenuated for virulence. To identify genes regulated by this TCSTS, we used RNA sequencing to compare the transcriptomes of the directed bvrS mutant and the wild-type parent; 104 genes were up-regulated and 22 down-regulated in the bvrS mutant. Amongst this set of differentially regulated genes we identified a number predicted to play a direct role in B. pseudomallei virulence. These included four genes involved in flagella biosynthesis, six in chemotaxis, six in phenylacetic acid (PA) catabolism, 19 involved in polyketide biosynthesis (four separate operons) and one encoding a thermolysin metalloprotease. We assayed the motility of the wild-type and mutant strains and the bvrS mutants demonstrated significantly reduced motility compared to the wild-type strain; complementation with intact bvrS restored motility. Furthermore, electron microscopy analysis indicated that the bvrS mutants produced highly reduced numbers of surface flagella. Therefore, BvrS is a component of a critical virulence-associated TCSTS that plays a vital role in regulating expression of flagellin synthesis and controlling motility.