Pseudomonas aeruginosa is a ubiquitous environmental bacterium and a clinically significant opportunistic human pathogen. Most significantly, P. aeruginosa infections are the primary cause of morbidity and mortality in individuals with the genetic disorder cystic fibrosis. P. aeruginosa has a large, ~6.3 Mbp genome, which enables adaptation to diverse environmental conditions via the production of a wide repertoire of nutrient sensors and molecular uptake mechanisms. Of these, the sensing and acquisition of the transition metal ion, zinc, is central to P. aeruginosa colonization. Bacterial zinc uptake predominantly occurs via ATP-binding cassette (ABC) transporters. Here we show that P. aeruginosa PAO1 acquires zinc via the ZnuABC ABC permease, in which ZnuA is the high affinity, zinc-specific binding protein. Metal content analysis of a P. aeruginosa ΔznuA mutant strain showed a 60% reduction in cellular zinc accumulation, while other metal ions were essentially unaffected. Despite the major reduction in zinc accumulation, minimal phenotypic differences were observed between the wild-type and ΔznuA mutant strains. Analysis of the P. aeruginosa PAO1 transcriptome under zinc limitation revealed significant changes in gene expression that enabled adaptation of the bacterium for growth under conditions of low zinc abundance. Forty-four genes were found to be up-regulated ≥ 4-fold under zinc limitation, including a number of novel putative zinc acquisition pathways and non-zinc-requiring paralogs of zinc-dependent proteins. Up-regulation of these genes was largely attributed to the Zinc uptake regulator, Zur. In this way, Zur regulates both zinc uptake and the magnitude of competition for the cellular Zn2+ pool, ensuring essential protein functions are maintained. Collectively, this study provides new insight into the acquisition of zinc by P. aeruginosa PAO1, revealing a hitherto unrecognized complexity in zinc homeostasis that enables the bacterium to survive under zinc limitation.