ABSTRACT Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A Δ plcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δ anr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence.