Iron is a critical nutrient for most microbial pathogens, and the innate immune system exploits this requirement by sequestering iron and other metals through a process termed nutritional immunity. The opportunistic pathogen Pseudomonas aeruginosa provides a model system for understanding the microbial response to host iron depletion, as this organism exhibits a high requirement for iron as well as an exquisite ability to overcome iron deprivation during infection. Hallmarks of P. aeruginosa’s iron starvation response include the induction of multiple high affinity iron acquisition systems and an “iron sparing response” that is post-transcriptionally mediated by the PrrF small regulatory RNAs (sRNAs). Here, we used liquid chromatography-tandem mass spectrometry to conduct label-free proteomics of the P. aeruginosa iron starvation response, revealing several iron-regulated processes that have not been previously described. Iron starvation induced multiple proteins involved in branched chain and aromatic amino acid catabolism, providing the capacity for iron-independent entry of carbons into the TCA cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were paradoxically increased, highlighting the central role of iron in P. aeruginosa metabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins, as well as the first experimental evidence for increased levels of twitching motility proteins upon iron starvation. Subsequent proteomics analyses demonstrated that the PrrF sRNAs were required for iron regulation of many of these newly-identified proteins, and we identified PrrF complementarity with mRNAs encoding key iron-regulated proteins involved in amino acid metabolism, iron-sulfur biogenesis, and zinc homeostasis. Combined, these results provide the most comprehensive view of the P. aeruginosa iron starvation response to date and outline novel roles for the PrrF sRNAs in the P. aeruginosa iron sparing response and pathogenesis.AUTHOR SUMMARYIron is central for the metabolism of almost all microbial pathogens, and as such this element is sequestered by the host innate immune system to restrict microbial growth. Defining the response of microbial pathogens to iron starvation is therefore critical for understanding how pathogens colonize and propagate within the host. The opportunistic pathogen Pseudomonas aeruginosa, which causes significant morbidity and mortality in compromised individuals, provides an excellent model for studying this response due to its high requirement for iron yet well-documented ability to overcome iron starvation. Here we used label-free proteomics to investigate the P. aeruginosa iron starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes are regulated through the iron responsive PrrF small regulatory RNAs, which are integral to P. aeruginosa iron homeostasis and virulence. These results demonstrate the power of proteomics for defining stress responses of microbial pathogens, and they provide the most comprehensive analysis to date of the P. aeruginosa iron starvation response.