Backgrounds <p>Pancreatic cancer is one of the most common malignancies in the gastrointestinal system, notorious for its high malignancy and low survival rate. Amentoflavone (AMF), an ingredient in various Traditional Chinese Medicines (TCM), possesses anticancer properties. However, the underlying mechanisms of its action remain unclear.&#xa0;</p> Methods <p>Initially, through the application of network pharmacology and molecular docking methodologies, we elucidated the putative core targets and signaling pathways underlying the antineoplastic potential of amentoflavone. Subsequently, we corroborated the therapeutic efficacy and underlying mechanisms of amentoflavone within both in vitro pancreatic cancer cell models and in vivo animal models.</p> Results <p>The key targets of amentoflavone were identified as SRC, AKT1, PI3KR1, VEGFA, ESR1, EGFR, and AR, among others. The mechanisms of action may involve protein phosphorylation, autophosphorylation, and positive regulation of apoptosis. KEGG analysis enrichment indicated that pathways such as Cancer, Estrogen Signaling, and PI3K/AKT/mTOR played a significant role. Molecular docking experiments demonstrated that amentoflavone showed good docking activity with the main target proteins. In vitro and in vivo experiments showed that amentoflavone effectively induced apoptosis in BxPC3 cells, inhibited cell migration, caused cell cycle arrest at the S phase, and regulated glycolysis in BxPC3 cells by inhibiting the PI3K/AKT/mTOR pathway. Additionally, amentoflavone effectively inhibited the growth and glycolytic process of pancreatic cancer xenografts in mice.</p> Conclusions <p>Our study elucidates that amentoflavone inhibits pancreatic cancer development by regulating the glycolysis process through the suppression of the PI3K/AKT/mTOR pathway, thereby providing novel targets and therapeutic strategies for the treatment of pancreatic cancer.</p>