Neonatal hypoxic-ischaemic encephalopathy (HIE) remains a leading cause of infant morbidity and mortality worldwide, with therapeutic hypothermia being the only clinically approved treatment. This study investigates the cortical expression pattern of neuroserpin during postnatal brain development and evaluates its neuroprotective potential in hypoxia-ischaemia (HI)-induced brain damage using a modified Rice-Vannucci model. Experiments were conducted in both male and female neuroserpin knockout (KO) mice and through administration of exogenous neuroserpin into the brain. Between postnatal day 4 to 14 (P4-P14), neuroserpin-immunoreactive cell density peaked at P8-P10 in cortical layers 5 and 6b, with a gradual increase in layers 2/3 and minimal changes in layers 4 and 6a. Despite comparable levels of ischaemic brain damage between the KO and wild-type (WT) mice, exogenous neuroserpin administration suppressed the HI-induced oxidative stress. Additionally, it reduced microglial activation and reactive astrogliosis in the cortex in mild HIE, mitigating cortical thinning and preserving neuronal distribution. These findings suggest that endogenous neuroserpin alone is insufficient for neuroprotection against HI-induced damage, but exogenous neuroserpin shows promise as a pharmacological intervention for mild neonatal HIE.