Reproductive function plays a central role in health but declines with aging. Recent studies have focused on natural flavonoids to mitigate reproductive aging, with the water extract of Ampelopsis grossedentata (WEA) showing promise due to its antioxidant, antitumor, antibacterial, and anti-inflammatory properties. However, the specific bioactive constituents and mechanisms of WEA in alleviating reproductive aging remain unclear. This experiment analyzed the active flavonoid components in WEA and predicted the molecular mechanism by which WEA alleviates reproductive system aging through network pharmacology. Furthermore, based on the predicted molecular mechanisms, the effects of WEA on laying hens' production performance, reproductive function, and intestinal health were explored. A total of 288 laying hens (55 weeks old) were assigned to four groups: control and three WEA doses (50, 150, 250 mg/kg). During the experiment, production performance indicators such as egg weight were recorded daily. After the 8-week period, biological samples were collected for analysis. Network pharmacology identified dihydromyricetin, myricetin, and (-)-epicatechin as key active components, primarily affecting the PI3K/AKT signaling pathway. WEA significantly improved egg quality, immune parameters, reproductive organ morphology, intestinal morphology, and serum sex hormone levels, and reduced inflammatory factor levels. WEA improved ovarian apoptosis by regulating the PI3K/AKT/mTOR pathway and alleviated oviduct inflammation by inhibiting the NF-κB pathway. WEA enhanced intestinal anti-inflammatory and antioxidant functions by regulating NF-κB and Nrf2 pathways and increased short-chain fatty acids in the hindgut. WEA altered the intestinal microbiome, particularly reducing the relative abundance of Methylobacterium-Methylorubrum in the foregut and Bacteroides in the hindgut. Correlation analysis revealed that WEA may alleviate oxidative and inflammatory responses by regulating intestinal microbiota, further impacting the PI3K/AKT cascade. In conclusion, WEA improves antioxidant, anti-inflammatory, and reproductive functions in laying hens by regulating the PI3K/AKT pathway and may alleviate oxidative and inflammatory responses by modulating microbiota. This study provides insights into the mechanism of WEA in improving reproductive performance and intestinal regulation.