Pseudomonas aeruginosa is a versatile opportunistic human pathogen that is able to colonize a broad spectrum of different aquatic and soil habitats. In the environment and during pathogenesis, P. aeruginosa encounters oxygen-limited and anaerobic environments. Particularly during chronic infection of the cystic fibrosis lung, oxygen-limiting conditions seem to contribute to persistent infection. Oxygen limitation increases antibiotic tolerance, robust biofilms and alginate biosynthesis, which contribute to the persistence of this opportunistic pathogen. Despite the importance of anaerobic metabolism during persistent infection of P. aeruginosa, we are just beginning to understand the underlying regulatory network and the molecular basis of how anaerobic metabolism contributes to a persistent infection. A deeper understanding of the anaerobic physiology of P. aeruginosa will allow the identification of new antibiotic targets and new therapeutic strategies.