Alzheimer's disease (AD) has an increasing prevalence, complicated pathogenesis and no effective cure. Emerging evidences show that flavonoid compounds such as xanthohumol (Xn) could play an important role as a dietary supplement or traditional Chinese herbal medicine in the management of diseases such as AD. This study aims to analyze the target molecules of Xn in the prevention and treatment of AD, and its potential mechanism from the perspective of metabolites. APP/PS1 mice 2- and 6-months old were treated with Xn for 3 months, respectively, the younger animals to test for AD-like brain disease prevention and the older animals to address therapeutic effects on the disease. Memantine (Mem) was selected as positive control. Behavioral tests were performed to assess the course of cognitive function. Urine samples were collected and analyzed by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) coupled with online Compound Discoverer software. Morris Water Maze (MWM) tests showed that Xn, like Mem, had a therapeutic but not a preventive effect on cognitive impairment. The expression levels of urinary metabolites appeared to show an opposite trend at different stages of Xn treatment, downregulated in the prevention phase while upregulated in the therapy phase. In addition, the metabolic mechanisms of Xn during preventive treatment were also different from that during therapeutic treatment. The signaling pathways metabolites nordiazepam and genistein were specifically regulated by Xn but not by Mem in the disease prevention stage. The signaling pathway metabolite ascorbic acid was specifically regulated by Xn in the therapeutic stage. In conclusion, dietary treatment with Xn altered the urinary metabolite profile at different stages of administration in APP/PS1 mice. The identified potential endogenous metabolic biomarkers and signal pathways open new avenues to investigate the pathogenesis and treatment of AD.