Background Innate immune responses of airway epithelium are important defences against respiratory pathogens and allergens. Newborn infants are at greater risk of severe respiratory infections compared to older infants. However, very little is known regarding human neonatal airway epithelium immune responses and whether age-related morphological and/or innate immune changes contribute to the development of airway disease. Methods We collected nasal epithelial cells from 41 newborn infants (23 term, 18 preterm) within 5 days of birth. Repeat sampling was achieved for 24 infants (13 term, 11 preterm) at a median age of 12.5 months. Morphologically and physiologically authentic well-differentiated primary paediatric nasal epithelial cell (WD-PNEC) cultures were generated and characterised using light microscopy and immunofluorescence. Results WD-PNEC cultures were established for 15/23 (65%) term and 13/18 (72%) preterm samples at birth, and 9/13 (69%) term and 8/11 (73%) preterm samples at one-year. Newborn and infant WD-PNEC cultures demonstrated extensive cilia coverage, mucous production and tight junction integrity. Newborn WD-PNECs took significantly longer to reach full differentiation and were noted to have much greater proportions of goblet cells compared to one-year repeat WD-PNECs. No differences were evident in ciliated/goblet cell proportions between term- and preterm-derived WD-PNECs at birth or one-year old. Conclusion WD-PNEC culture generation from newborn infants is feasible and represents a powerful and exciting opportunity to study differential innate immune responses in human airway epithelium very early in life.