Supplemental oxygen is often used to treat neonates with respiratory disorders. Preclinical studies have demonstrated that neonatal hyperoxia injures the distal small intestine and activates nuclear factor-κB (NF-κB). Cathelicidin inhibits NF-κB activity and ameliorates lipopolysaccharide-induced intestinal barrier disruption in rats. Sprague-Dawley rat pups were reared in either room air (RA) or hyperoxia (85% O) and were randomly treated with low-dose cathelicidin (4 mg/kg, LDC) and high-dose cathelicidin (HDC, 8 mg/kg) in 0.05 mL of normal saline (NS) administered intraperitoneally on postnatal days 1-6. The following six groups were obtained: RA + NS, RA + LDC, RA + HDC, O + NS, O + LDC, and O + HDC. The animals were sacrificed and the terminal ileum was removed for Western blot and histological analyses on postnatal day 7. The hyperoxia-reared rats exhibited significantly lower body weights, higher intestinal injury scores, lower occludin and ZO-1 expression, higher intestinal permeability and inducible IκB kinase inhibitor (IKKi) and NF-κB expression than the RA-reared rats. Cathelicidin treatment attenuated intestinal injury as evidenced by lower intestinal injury scores and intestinal permeability and higher intestinal barrier protein expression. The decrease in intestinal injury was accompanied by a decrease in IKKi and NF-κB. Cathelicidin attenuated hyperoxia-induced intestinal injury in the newborn rats, likely through NF-κB activity inhibition.