Macrolides are frequently prescribed antibiotics, used to treat a spectrum of respiratory and skin infections. They are also known for their off-label use, some macrolides being effective against inflammation and oxidative stress, affecting several signaling pathways. Recently, their enhancing effects on the respiratory epithelial barrier have become more evident. In this study the non-antimicrobial effects of the traditional 14-membered macrolide antibiotics; erythromycin (ERY), clarithromycin (CLARI) and roxithromycin (ROXI), the 15-membered nitrogen containing macrolide, azithromycin (AZM) and the ketolide solithromycin (SOLI) were analyzed in vitro. A bronchial epithelial cell line, VA10, was treated with macrolides for 14 and 21 days in air-liquid interface (ALI) condition. Along with RNA sequencing, transepithelial electrical resistance (TEER), permeability and histological effects were observed to evaluate the epithelial barrier. Treatment with AZM differs from the other macrolides by a more pronounced increase in TEER, thickness of cell layer, decreased permeability and increased accumulation of phospholipids. ERY and CLARI showed both moderate increase in TEER and decreased permeability. Collectively, we have shown that macrolides have profound effects on gene expression and phenotype in human bronchial cells. AZM has distinctly greater epithelial barrier-inductive properties, although all macrolides tested affect gene expression, particularly gene sets involving inflammation, lipid metabolism and oxidative stress.