BACKGROUND/AIM: Biological aging refers to the progressive deterioration of an organism's functions due to accumulated cellular and molecular damage. NeuroD2, a critical transcription factor, plays a crucial role in neuronal development and synaptic maturation. This study investigates the role of NeuroD2 in hippocampal neuron organization, focusing on hilar mossy cells during aging, and employs bioinformatics to identify NeuroD2 targets linked to cellular aging. MATERIALS AND METHODS: We used 21 adult C57Bl/6 mice, categorized into three age groups: young (2 months), old (12 months), and very old (24 months). Mice were administered BrdU intraperitoneally for three consecutive days, 10 days before euthanasia. The brains were harvested and analyzed using immunofluorescence, Western blotting, and quantitative real-time PCR to evaluate NeuroD2 expression and associated signaling pathways in hippocampal tissue. Additionally, bioinformatics analysis was performed using two open-access datasets (ProteomeXchange Consortium, Dataset identifier: PXD043352; Gene Expression Omnibus, GEO: GSE67539) and to explore potential interactions between NeuroD2 targets and biological aging components. RESULTS: Our results revealed that NeuroD2 expression changed significantly with age and correlated with AKT signaling. Moreover, we identified a potential link between NeuroD2 activity and the AKT pathway, indicating NeuroD2's role in mitigating aging-related stress. Bioinformatics analysis identified 513 down-regulated and 638 up-regulated proteins, with NeuroD2 targets involved in axonal projection showing increased expression in older mice. Contrary to expectations, pathway enrichment analysis highlighted axon guidance molecules rather than oxidative stress markers. CONCLUSION: This study underscores NeuroD2's critical role in preserving hippocampal integrity and reveals molecular mechanisms underlying age-related neuronal changes. Our findings provide insights into the role of NeuroD2 in regulating key pathways during healthy aging, potentially mitigating the impacts of aging.