Previously, we identified the uncharacterized predicted membrane protein PA2663 of Pseudomonas aeruginosa PAO1 as a virulence factor using a poplar tree model; PA2663 was induced in the poplar rhizosphere and, upon inactivation, it caused 20-fold lower biofilm formation (Attila et al., Microb Biotechnol, 2008). Here, we confirmed that PA2663 is related to biofilm formation by restoring the wild-type phenotype by complementing the PA2663 mutation in trans and investigated the genetic basis of its influence on biofilm formation through whole-transcriptome and -phenotype studies. Upon inactivating PA2663 by transposon insertion, the psl operon that encodes a galactose- and mannose-rich exopolysaccharide was highly repressed (verified by RT-PCR). The inactivation of PA2663 also repressed 13 pyoverdine genes, which eliminated the production of the virulence factor pyoverdine in P. aeruginosa. The inactivation of PA2663 also affected other quorum-sensing-related phenotypes in that it repressed the Pseudomonas quinolone signal (PQS) genes, which abolished PQS production, and repressed lasB, which decreased elastase activity sevenfold. Genes were also induced for motility and attachment (PA0499, PA0993, PA2130, and PA4549) and for small molecule transport (PA0326, PA1541, PA1632, PA1971, PA2214, PA2215, PA2678, and PA3407). Phenotype arrays also showed that PA2663 represses growth on D-gluconic acid, D-mannitol, and N-phthaloyl-L-glutamic acid. Hence, the PA2663 gene product increases biofilm formation by increasing the psl-operon-derived exopolysaccharides and increases pyoverdine synthesis, PQS production, and elastase activity while reducing swarming and swimming motility. We speculate that PA2663 performs these myriad functions as a novel membrane sensor.