<jats:title>ABSTRACT</jats:title> <jats:p> Most <jats:italic>Burkholderia pseudomallei</jats:italic> strains are intrinsically aminoglycoside resistant, mainly due to AmrAB-OprA-mediated efflux. Rare naturally occurring or genetically engineered mutants lacking this pump are aminoglycoside susceptible despite the fact that they also encode and express BpeAB-OprB, which was reported to mediate efflux of aminoglycosides in the Singapore strain KHW. To reassess the role of BpeAB-OprB in <jats:italic>B. pseudomallei</jats:italic> aminoglycoside resistance, we used mutants overexpressing or lacking this pump in either AmrAB-OprA-proficient or -deficient strain 1026b backgrounds. Our data show that BpeAB-OprB does not mediate efflux of aminoglycosides but is a multidrug efflux system which extrudes macrolides, fluoroquinolones, tetracyclines, acriflavine, and, to a lesser extent, chloramphenicol. Phylogenetically, BpeAB-OprB is closely related to <jats:italic>Pseudomonas aeruginosa</jats:italic> MexAB-OprM, which has a similar substrate spectrum. AmrAB-OprA is most closely related to MexXY, the only <jats:italic>P. aeruginosa</jats:italic> efflux pump known to extrude aminoglycosides. Since BpeAB-OprB in strain KHW was also implicated in playing a major role in export of acylated homoserine lactone (AHL) quorum-sensing molecules and in expression of diverse virulence factors, we explored whether this was also true in the strain 1026b background. The results showed that BpeAB-OprB was not required for AHL export, and mutants lacking this efflux system exhibited normal swimming motility and siderophore production, which were severely impaired in KHW <jats:italic>bpeAB-oprB</jats:italic> mutants. Biofilm formation was impaired in 1026b Δ( <jats:italic>amrRAB-oprA</jats:italic> ) and Δ( <jats:italic>amrRAB-oprA</jats:italic> ) Δ( <jats:italic>bpeAB-oprB</jats:italic> ) mutants. At present, we do not know why our BpeAB-OprB susceptibility and virulence factor expression results with 1026b and its derivatives are different from those previously published for Singapore strain KHW.