AbstractObjectivePrevious in vitro transcriptomic profiling suggests azithromycin exerts its effects in patients with chronic rhinosinusitis (CRS) via modulation of type 1 inflammation and restoration of epithelial barrier function. We wished to verify these postulated effects using in vitro models of epithelial repair and in vivo transcriptional profiling.Study DesignFunctional effects of azithromycin in CRS were verified using in vitro models of wounding. The mechanism of the effect of azithromycin was assessed in vivo using transcriptomic profiling.SettingAcademic medical center.MethodsEffects of azithromycin on the speed of epithelial repair were verified in a wounding model using primary nasal epithelial cells (pNEC) from CRS patients. Nasal brushings collected pre‐and posttreatment during a placebo‐controlled trial of azithromycin for CRS patients unresponsive to surgery underwent transcriptomic profiling to identify implicated pathways.ResultsAdministration of azithromycin improved the wound healing rates in CRS pNECs and prevented the negative effect of Staphylococcus aureus on epithelial repair. In vivo, response to azithromycin was associated with downregulation in pathways of type 1 inflammation, and upregulation of pathways implicated in the restoration of the cell cycle.ConclusionRestoration of healthy epithelial function may represent a major mode of action of azithromycin in CRS. In vitro models show enhanced epithelial repair, while in vivo transcriptomics shows downregulation of pathways type 1 inflammation accompanied by upregulation of DNA repair and cell‐cycle pathways. The maximal effect in patients with high levels of type 1‐enhanced inflammation suggests that azithromycin may represent a novel therapeutic option for surgery‐unresponsive CRS patients.