Nitric oxide synthase 2 (NOS2) is expressed in human respiratory epithelium and is induced in inflammatory airway diseases. However, relatively little is known about the mechanisms underlying the regulation of NOS2 activity or the targets of NOS2-derived NO in this cell-type. We hypothesized that the large-scale identification of NOS2-protein interactions, and quantification of changes to the NOS2 “interactome” in response to inflammatory stimuli, would provide new insights into the function and regulation of NOS2. We overexpressed Flag-tagged NOS2 in airway epithelial (A549) cells and used mass spectrometry to identify NOS2 co-immunoprecipitating proteins. We further utilized stable isotope labeling of amino acids in cell culture (SILAC) to quantify the coordinate effects of cytokine stimulation on these interactions. Multi-protein networks dominated the NOS2 interactome, and inducible interactions with allosteric activators and with the ubiquitin–proteasome system were correlated with cytokine-dependent increases in NO metabolites and in NOS2 ubiquitination, respectively. We also identified a novel NOS2-interacting protein and associated ubiquitin ligase complex that may be one of several regulators of NOS2 stability in the A549 cell. We will discuss the significance of these findings and the applicability of these proteomic techniques for studying NOS2 in other cellular systems.