AbstractUnderstanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.