Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the HA of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropism. In accordance with distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and non-ciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected non-ciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, non-ciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C, however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression of immune mediator genes and resulted in more cell damage/loss when compared with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE: Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.