The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100,000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix-turn-helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)-PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at -127 and -88 bp upstream of their initiation codons with Shine-Dalgarno and putative promoter sequences containing -10 and -35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of P. aeruginosa in chronic lung infection.