Despite widely known detrimental effects on the developing brain, supplemental oxygen is still irreplaceable in the management of newborn infants with respiratory distress. Identifying downstream mechanisms underlying oxygen toxicity is a key step for development of new neuroprotective strategies. Main purpose of this study is to investigate whether NLRP3 inflammasome activation has a role in the pathogenesis of hyperoxia-induced preterm brain injury. C57BL6 pups were randomly divided into either a hyperoxia group (exposed to 90 % oxygen from birth until postnatal day 7) or control group (maintained in room air; 21 % O-2). At postnatal day 7, all animals were sacrificed. Immunohistochemical examination revealed that hyperoxic exposure for seven days resulted in a global increase in NLRP3 and IL-1 beta immunopositive cells in neonatal mouse brain (p = 0.001). There was a significant rise in Caspase-1 positive cell count in prefrontal and parietal area in the hyperoxia group when compared with controls (p = 0.001). Western blot analysis of brain tissues showed elevated NLRP3, IL-1 beta and Caspase-1 protein levels in the hyperoxia group when compared with controls (p = 0.001). To the best of our knowledge, this is the first study that investigates an association between hyperoxia and establishment of NLRP3 inflammasome in preterm brain.