Bats are natural reservoir hosts for a variety of viruses including SARS-CoV, Ebola, Hendra (HeV), Nipah (NiV), and Marburg which are highly pathogenic in humans and susceptible animals[1-5], yet rarely cause clinical disease in bats. The Australian black flying fox (Pteropus alecto) is no exception, having been identified as a natural host for a number of viruses including HeV which causes fatal disease in humans and horses[6,7]. Therefore, decoding bats' ability to control virus infection has the potential to inform the development of interventions to prevent spillover of viruses to other susceptible hosts and identify new therapeutic targets and vaccines. Previous studies to characterise the host-pathogen responses of bats have predominantly used 2D cell culture systems consisting of a monolayer of cells. In recent years, more complex cell culture systems, including organoids and transwell models have become commonplace for studies of humans and other species, providing more physiologically relevant in vitro systems. A few physiologically relevant 3D culture models have been established for other bat species to understand their antiviral capabilities[8,9] but no ex vivo models have been reported for P. alecto. We isolated airway epithelial cells from P. alecto tracheobronchial tissues and then differentiated them at the air-liquid interface to generate pseudostratified 3D airway epithelium cultures. The bat epithelial cell model exhibited a mucociliary phenotype, characterised by mucus secretion and beating cilia on the surface of ciliated cells. Histological staining confirmed that the bat tracheal cells formed an epithelium consisting of basal, goblet, club and ciliated cells. Here we describe the characteristics of this model, including the innate immune response and demonstrate that bat epithelial cells provide a physiologically relevant model for studying the host-pathogen response of P. alecto.