Background: Continuous positive airway pressure (CPAP) is a primary form of respiratory support used in the intensive care of preterm infants, but its long-term effects on airway (AW) function are unknown. Methods: We developed a neonatal mouse model of CPAP treatment to determine whether it modifies later AW reactivity. Unanesthetized spontaneously breathing mice were fitted with a mask to deliver CPAP (6 cmH₂O, 3 h/day) for 7 consecutive days starting at postnatal day 1. AW reactivity to methacholine was assessed using the in vitro living lung slice preparation. Results: One week of CPAP increased AW responsiveness to methacholine in male, but not female mice, compared to untreated control animals. The AW hyper-reactivity of male mice persisted for 2 wk (at P21) after CPAP treatment ended. Four days of CPAP, however, did not significantly increase AW reactivity. Females also exhibited AW hyper-reactivity at P21, suggesting a delayed response to early (7 d) CPAP treatment. The effects of 7 d of CPAP on hyper-reactivity to methacholine were unique to smaller AWs whereas larger ones were relatively unaffected. Conclusion: These data may be important to our understanding of the potential long-term consequences of neonatal CPAP therapy used in the intensive care of preterm infants.