Although periventricular white matter injury is a leading cause of major neurologic disability in premature infants, the relationship between myelination deficiency and long-term cognitive dysfunction is not well understood. The purpose of this study was to investigate oligodendrocytes myelination and long-term spatial cognitive function in rats with perinatal hypoxia-ischemia (HI). Postnatal day 3 (P3) rats were subjected to right carotid artery ligation followed by 2.5 h of hypoxia (6% oxygen). Brain injury during the early and late phases was evaluated by immunostaining at P6 (72 h after the injury) and P47. Spatial cognitive function was evaluated at P42 using the Morris Water Maze test followed by histologic evaluation. HI caused an increase in pre-oligodendrocytes, astrocytes, and microglia in the ipsilateral white matter 72 h after the insult compared to contralateral regions and sham-operated controls (both p<0.05). There were significant decreases in myelin basic protein (MBP)and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-labeled oligodendrocytes with glial fibrillary acidic protein (GFAP)-labeled glial scarring in the ipsilateral periventricular white matter at P47 compared to contralateral regions and sham-operated controls (all p<0.05). The rats with HI had spatial learning deficits in navigation trials (longer escape latency and swimming distance) and memory dysfunction in probe trials (fewer number of platform crossings and percentage of time in the target quadrant) compared with sham-operated controls (p<0.05). In this neonatal rat model of HI, myelination deficiency induced by activated astrocytes and microglia during the early phase with subsequent glial scarring was associated with long-term spatial learning and memory dysfunction.