© 2019 by The American Association of Immunologists, Inc. Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, antiinflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-kB and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-g, IL-4/-13, or with azithromycin plus IFN-g) and stimulated with LPS. The effect of azithromycin on NF-kB and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug’s effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-kB activation by decreasing p65 nuclear translocation, although blunting the degradation of IkBa was due, at least in part, to a decrease in IKKb kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKKb protein expression. Moreover, incubation with the IKKb inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-kB signaling pathways through the drug’s effect on p65 nuclear translocation and IKKb.