The quorum sensing (QS) signalling system of Pseudomonas aeruginosa controls many important functions, including virulence. Although the production of the QS signal molecule N-3-oxo-dodecanoyl-homoserine lactone (3OC(12)-HSL) is positively autoregulated, its concentration reaches a steady level long before stationary phase. The RsaL protein represses transcription of the lasI signal synthase gene, and thus reduces QS signal production. We show that RsaL binds simultaneously with LasR to the rsaL-lasI bidirectional promoter thereby preventing the LasR-dependent activation of both genes. In an rsaL mutant, 3OC(12)-HSL production continues to increase throughout growth. Thus RsaL provides homeostasis by functioning in opposition to LasR and limiting 3OC(12)-HSL production to a physiological concentration. Furthermore, transcription profiling revealed that RsaL regulates 130 genes independent of its effect on QS signal molecule production, including genes involved in virulence. We show that RsaL can repress pyocyanin and hydrogen cyanide virulence genes in two ways: directly, by binding to their promoters, and indirectly, by decreasing levels of the signals for their QS signal-dependent transcription. These investigations highlight the importance of RsaL as a global regulator of P. aeruginosa physiology that provides a counterbalance to 3OC(12)-HSL-dependent gene activation via multiple mechanisms.