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Background SmeYZ is a constitutively expressed efflux pump in Stenotrophomonas maltophilia. Previous studies demonstrated that: (i) smeYZ inactivation causes compromised swimming, oxidative stress tolerance and aminoglycoside resistance; and (ii) the ΔsmeYZ -mediated pleiotropic defects, except aminoglycoside susceptibility, result from up-regulation of entSCEBB′FA and sbiAB operons, and decreased intracellular iron level. Objectives To elucidate the modulatory role of SmeQ, a novel cytoplasmic protein, in ΔsmeYZ -mediated pleiotropic defects. Methods The presence of operons was verified using RT–PCR. The role of SmeQ in ΔsmeYZ -mediated pleiotropic defects was assessed using in-frame deletion mutants and functional assays. A bacterial adenylate cyclase two-hybrid assay was used to investigate the protein–protein interactions. Gene expression was quantified using quantitative RT–PCR (RT–qPCR). Results SmeYZ and the downstream smeQ formed an operon. SmeQ inactivation in the WT KJ decreased aminoglycoside resistance but did not affect swimming and tolerance to oxidative stress or iron depletion. However, smeQ inactivation in the smeYZ mutant rescued the ΔsmeYZ -mediated pleiotropic defects, except for aminoglycoside susceptibility. In the WT KJ, SmeQ positively modulated SmeYZ pump function by transcriptionally up-regulating the smeYZQ operon. Nevertheless, in the smeYZ mutant, SmeQ exerted its modulatory role by up-regulating entSCEBB′FA and sbiAB operons, decreasing intracellular iron levels, and causing ΔsmeYZ -mediated pleiotropic defects, except for aminoglycoside susceptibility. Conclusions SmeQ is the first small protein identified to be involved in efflux pump function in S. maltophilia. It exerts modulatory effect by transcriptionally altering the expression of target genes, which are the smeYZQ operon in the WT KJ, and smeYZQ , entSCEBB′FA and sbiAB operons in smeYZ mutants. [ABSTRACT FROM AUTHOR]