Despite improvements in therapy, HIV‐1 infection still causes serious lung infections and respiratory failure. Many of the manifestations of HIV‐1 infection are mediated by viral‐related proteins, such as gp120 and Tat, and not by direct infection of cells. Tight junction (TJ) protein structure and function is associated with HIV‐1‐related breakdown of the blood‐brain barrier, but to date this has not been examined in the lung. We used the hemizygous NL4‐3Δgag/pol transgenic rat to test the hypothesis that HIV‐1‐related proteins interfere with alveolar epithelial TJ assembly and barrier formation. We assessed alveolar epithelial barrier function both in vivo and in primary alveolar epithelial monolayers in vitro. Transgenic HIV‐1 expression significantly decreased lung liquid clearance in vivo and decreased alveolar epithelial monolayer integrity in vitro. In parallel, HIV‐1 transgenic expression significantly decreased the TJ proteins claudin‐4, claudin‐18, occludin and ZO‐1 by western blot analysis, and fluorescence staining showed that ZO‐1 and occludin protein localization to alveolar epithelial cell junctions was abnormal. Our findings suggest that HIV‐1‐related proteins interfere with tight junction assembly and function within the alveolar epithelium, and this could explain in part why HIV‐1‐infected individuals are at higher risk for respiratory failure.