Background: Azithromycin polarizes macrophages into an alternative phenotype and promotes a regulated immunity. Arginase is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. Hypothesis: Decreases in inflammation in response to Pseudomonas aeruginosa (PA) pneumonia achieved by polarizing macrophages to an alternative phenotype is dependent upon the production of arginase. Methods: Requirement of arginase was examined by pharmacological inhibition using S-(2-boronoethyl)- l-cysteine (BEC) or l-norvaline and by infecting arginase-1 conditional knock-out mice (Arg1flox/flox;Lyz2-cre (Arg1Δm)) with PA intratracheally. Arg1ΔM and control Arg1flox/flox mice were then dosed with azithromycin daily via oral gavage beginning four days prior to infection. Analysis of weight loss in addition to characterization of inflammatory cells and cytokine production via flow cytometry was performed. Macrophages were then stimulated with LPS and polarized with IL4/13, IFNγ, or azithromycin plus IFNγ. Western blot for signaling mediators, p65 translocation assay, and immunofluorescence were performed. Results: Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory response compared to the Arg1flox/flox mice. Arg1Δm mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to the Arg1flox/flox mice. Conversely, global arginase inhibition resulted in greater weight loss along with greater neutrophil and macrophage infiltration compared to Arg1Δm mice. BEC and l-norvaline treated mice had higher numbers of lymphocytes in their lymph nodes with variable effects on airway lymphocyte counts. Azithromycin treatment comparably reduced the acute inflammatory responses in both Arg1Δm and Arg1flox/flox mice. To evaluate this mechanism, we show in vitro that azithromycin decreases NF-κB activation by preventing p65 nuclear translocation and by decreasing STAT1 activation in a concentration-dependent manner. These effects were reversed with IKKβ inhibition. Conclusions: Myeloid arginase is essential for control of inflammatory responses in PA pneumonia with potentially different effects of other cellular sources demonstrated with global arginase inhibition. Azithromycin reduces excessive inflammation even in the absence of arginase, potentially through a cross-inhibitory mechanism involving STAT1 and NF-κB pathways through IKKβ.