Brain injury in preterm infants is a major cause of disability and mortality in children. GSK-3β is a common pathogenic factor for cognitive dysfunction and involves in neuronal proliferation and differentiation. However, GSK-3β affected neuronal differentiation and its molecular pathogenesis after hypoxic-ischemic brain damage in neonatal rats remains unclear. This study investigated the effects of GSK-3β inhibitor (TWS119) on cell cycle regulatory proteins, a neuronal differentiation factor (CEND1), maturation neurons, T-box brain transcription factor 1 (TBR1)-positive neurons to clarify the mechanisms of hypoxic-ischemic brain damage in neonatal rats. We used hypoxic-ischemic Sprague-Dawley neonatal rats with brain damage as models. These rats were used for investigating the effect of GSK-3β on cell cycle regulatory proteins, neuronal differentiation factor (CEND1), maturation neurons, TBR1-positive neurons by western blot and immunofluorescence. Cyclin D1 (a positive cell cycle regulator) expression decreased, and p21 (a negative cell cycle regulator) expression increased in the TWS119 group compared to the hypoxia-ischemia (HI) group 7 days after HI. Additionally, compared to the HI group, TWS119 treatment up-regulated CEND1 expression and promoted neuronal differentiation and cortex development based on NeuN and TBR1 expression. Our study suggests that the GSK-3β inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats by inhibiting cell cycle pathway.