To observe the effects of Huannao Yicong Formula (e, HYF) on learning and memory and it's regulating effect on gamma-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer's disease. Sixty APP/PS1 transgenic mice, randomly allocated into 4 groups, the model group, the donepezil group (0.65 mg/kg), HYF low-dose group (HYF-L, 5.46 g/kg) and HYF high-dose group (HYF-H, 10.92 g/kg), 15 for each group. Another 15 C57BL/6J mice with the same age and same genetic background were allocated into the control group, proper dosage of drugs or distilled water were given by intragastric administration once daily for 12 weeks. After 12 weeks of administration, the learning and memory abilities of mice in each group was evaluated by the morris water maze test, amyloid precursor protein (APP), A beta(1-40) and A beta(1-42) levels in hippocampus were detected by enzyme-linked immunosorbent assay, gamma-secretase was detected by dual luciferase assaying, the levels of APH-1a, hypoxia-inducible factor 1 alpha (HIF-1 alpha), cAMP response element-binding protein (CREB) and PEN-2 and their mRNA expression was measured by Western blot and real-time polymerase chain reaction. HYF can ameliorate learning and memory deficits in APP/PS1 transgenic mice by decreasing the escape latency, improving the number of platform crossing and swimming speed (P < 0.01, P < 0.05). HYF can decrease the levels of APP, A beta(1-40), A beta(1-42) and the activity of gamma-secretase in hippocampus of Alzheimer's disease model mice. HYF can down-regulate the levels of CREB and PEN-2 and the expression of their mRNA. HYF can improve the learning and memory ability by inhibiting the activity of gamma-secretase through the CREB/PEN-2 signaling pathway, and this may be one of the therapeutic mechanisms of HYF in Alzheimer's disease.