Although endoscopic studies in adult humans have suggested that laryngeal closure can limit alveolar ventilation during nasal intermittent positive pressure ventilation (nIPPV), there are no available data regarding glottal muscle activity during nIPPV. In addition, laryngeal behavior during nIPPV has not been investigated in neonates. The aim of the present study was to assess laryngeal muscle response to nIPPV in nonsedated newborn lambs. Nine newborn lambs were instrumented for recording states of alertness, electrical activity [electromyograph (EMG)] of glottal constrictor (thyroarytenoid, TA) and dilator (cricothyroid, CT) muscles, EMG of the diaphragm (Dia), and mask and tracheal pressures. nIPPV in pressure support (PS) and volume control (VC) modes was delivered to the lambs via a nasal mask. Results show that increasing nIPPV during wakefulness and quiet sleep led to a progressive disappearance of Dia and CT EMG and to the appearance and subsequent increase in TA EMG during inspiration, together with an increase in trans-upper airway pressure (TUAP). On rare occasions, transmission of nIPPV through the glottis was prevented by complete, active glottal closure, a phenomenon more frequent during active sleep epochs, when irregular bursts of TA EMG were observed. In conclusion, results of the present study suggest that active glottal closure develops with nIPPV in nonsedated lambs, especially in the VC mode. Our observations further suggest that such closure can limit lung ventilation when raising nIPPV in neonates.