Recently, gintonin and gintonin-enriched fraction (GEF) have been isolated from ginseng, a herbal medicine. Gintonin induces [Ca2+]i transition in cultured hippocampal neurons and stimulates acetylcholine release through LPA receptor activation. Oral administration of GEF is linked to hippocampus-dependent cognitive enhancement and other neuroprotective effects; however, effects of its long-term administration on hippocampal gene expression remains unknown. Here, we used next-generation sequence (NGS) analysis to examine changes in hippocampal gene expressions after long-term oral administration of GEF. C57BL/6 mice were divided into three groups: control group, GEF50 (GEF 50 mg/kg, p.o.), and GEF100 (GEF 100 mg/kg, p.o.). After 22 days, total RNA was extracted from mouse hippocampal tissues. NGS was used for gene expression profiling; quantitative-real-time PCR and western blot were performed to quantify the changes in specific genes and to confirm the protein expression levels in treatment groups. NGS analysis screened a total of 23,282 genes, analyzing 11-related categories. We focused on the neurogenesis category, which includes four genes for candidate markers: choline acetyltransferase (ChAT) gene, β3-adrenergic receptor (Adrb3) gene, and corticotrophin-releasing hormone (Crh) gene, and tryptophan 2,3-dioxygenase (Tdo2) gene. Real-time PCR showed a marked overexpression of ChAT, Adrb3, and Crh genes, while reduced expression of Tdo2. Western blot analysis also confirmed increased ChAT and decreased Tdo2 protein levels. We found that GEF affects mouse hippocampal gene expressions, associated with memory, cognitive, anti-stress and anti-anxiety functions, and neurodegeneration at differential degree, that might explain the genetic bases of GEF-mediated neuroprotective effects. © 2020 Korea Institute of Oriental Medicine. Publishing services by Elsevier B.V.