Background: Osteopontin (OPN) is a highly phosphorylated sialoprotein and a soluble cytokine that is widely expressed in a variety of tissues, including the brain. OPN and OPN-derived peptides have been suggested to have potential neuroprotective effects against ischemic brain injury, but their role in preterm brain injury is unknown. Methods: We used a hypoxia-ischemia (HI)-induced preterm brain injury model in postnatal day 5 mice. OPN and OPN-derived peptides were given intracerebroventricularly and intranasally before HI. Brain injury was evaluated at 7 days after the insults. Results: There was a significant increase in endogenous OPN mRNA and OPN protein in the mouse brain after the induction of HI at postnatal day 5. Administration of full-length OPN protein and thrombin-cleaved OPN did not affect preterm brain injury. This was demonstrated with both intracerebroventricular and intranasal administration of OPN as well as in OPN-deficient mice. Interestingly, both N134-153 and C154-198 OPN-derived peptides increased the severity of brain injury in this HI-induced preterm brain injury model. Conclusions: The neuroprotective effects of OPN are age-dependent, and, in contrast to the more mature brain, OPN-derived peptides potentiate injury in postnatal day 5 mice. Intranasal administration is an efficient way of delivering drugs to the central nervous system (CNS) in neonatal mice and is likely to be an easy and noninvasive method of drug delivery to the CNS in preterm infants.