alpha-Hederin is a pentacyclic triterpenoid saponin extracted from Pulsatilla chinensis, which is known to suppress cancer cell proliferation. However, the role of this compound in pancreatic cancer cells remains unclear. The aim of this study was to reveal the docking molecular and the regulatory mechanism of alpha-hederin in pancreatic cancer. Here, we cultured Capan-1 and BxPC-3 cells and treated with different doses of alpha-hederin. Cell proliferation, migration, and apoptosis were detected using CCK8, EdU, Transwell, wound healing assay, and flow cytometer apoptosis assay. The in vivo experiment using subcutaneous tumor and caudal vein metastasis model to evaluate the inhibit effect of alpha-hederin Capan-1 cell tumor growth and metastasis. Proteomics were used to reveal the regulatory mechanism. The result shows that alpha-hederin treatment inhibits cell proliferation and invasion in concentration dependence way in both vivo and in vitro. The result shows that the IC50 for both Capan-1 and BxPC-3 were 32.5 M mu and 15 M mu, respectively. Flow cytometer apoptosis assay shows that alpha-hederin treatment promotion cell apoptosis in both Capan-1 and BxPC-3 cells. Proteomics and immunofluorescence detection confirmed that alpha-hederin treatment downregulated lactate dehydrogenase A (LDHA) expression and inhibited glycolysis. Molecular docking of alpha-hederin and LDHA proteins further confirmed that LDHA is a target of alpha-hederin. Taken together, this study confirms that alpha-hederin inhibits pancreatic cancer cell proliferation and invasion by inhibiting LDHA-mediated glycolysis. LDHA may be a direct target of alpha-hederin in pancreatic cancer.