Hippocampal cholinergic neurostimulating peptide (HCNP) was originally discovered from soluble fraction of rat hippocampus, which induces acetylcholine synthesis in the medial septal nucleus. HCNP is aligned at the N-terminal region of 21-kD HCNP precursor protein (HCNP-pp). We reported that HCNP-pp knockout (KO) mice may be an adequate model for cholinergic functional impairment in septo-hippocampal interactions. Here, to encourage drug discovery improving clinical symptom for Alzheimer’s disease we tried to generate a new model mice with cholinergic dysfunction and amyloid pathogenesis. The HCNP-pp KO mice was crossed with Amyloid precursor protein knock-in mice with three pathogenic mutation (AppNL-G-F KI). The mice was evaluated by the behavioral function, and electro-physiological phenotype by theta power-related cholinergic function and fEPSP during long-term potentiation. Some molecules-associated with the cholinergic/ glutamatergic terminals, amyloid pathogenesis or inflammation reaction was also examined by western blot and immunohistochemistry. The memory impairment became evident from earlier period, 9-momths old, in HCNP-pp KO×App KI mice than App KI mice, 12-momths old. Reductions of both the theta power and fEPSP were also confirmed in the hippocampus of HCNP-pp KO×App KI mice compared to App KI mice. However, no significant difference in molecules-associated with amyloid pathogenesis and inflammation reaction was revealed between two groups. Those results suggest that the cholinergic dysfunction thorough HCNP-pp KO may phenotypically accelerate impairment of cognitive function in App KI mice. The new model mice could be utilized as a pathological animal model with cholinergic dysfunction and amyloid pathogenesis for drug discovery against Alzheimer’s disease.