Neonatal hypoxia-ischemia (H-I) is a common cause of perinatal morbidity and mortality leading to prominent activation of caspase-3 in the brain. Previous studies have shown that acute inhibition of caspase-3 can protect against neonatal H-I in rats. In this study, we investigated brain injury following neonatal H-I in mice deficient in caspase-3. Wild-type, caspase-3+/- and caspase-3-/- mice underwent unilateral carotid ligation at postnatal day (P) 7, followed by 45 min of exposure to 8% oxygen. Surprisingly, tissue loss at P14 was significantly higher in caspase-3-/- mice when compared to wild-type littermates. As in rats, we found that acute inhibition of caspase-3 in mice leads to decrease in tissue loss at P14. There was no difference in nuclear morphology, DNA laddering or calpain activation between caspase-3-/-caspase-3+/- and wild-type mice subjected to H-I, and there was no evidence for compensatory activation of other caspases in caspase-3-/- mice. Also, all genotypes showed evidence of mitochondrial dysfunction after H-I, suggesting that this is a critical point in regulation of neuronal cell death following neonatal H-I. Our results suggest that long-term inhibition of caspase-3 during development, unlike acute inhibition, leads to upregulation of caspase-3-independent cell death pathways and increases the vulnerability of the developing brain to neonatal H-I injury.