Abstract: This study investigated the antitumor activity and molecular mechanisms of penfluridol (PF), an antipsychotic drug, in gastric cancer treatment. In vitro assays including CCK-8, colony formation, wound healing, Transwell migration, Hoechst 33342 staining, and flow cytometry demonstrated that penfluridol significantly inhibited proliferation and migration of SGC-7901 and HGC-27 gastric cancer cells in a concentration-dependent manner, while inducing apoptosis. Transcriptomic profiling combined with molecular docking revealed stable binding conformations between penfluridol and heat shock protein 90 (HSP90) or mouse double minute 2 (MDM2). Western blot analysis further confirmed that penfluridol suppressed the HSP90/protein kinase B/MDM2/tumor protein p53 (HSP90/Akt/MDM2/p53) pathway, characterized by decreased HSP90 expression and concomitant upregulation of p53 protein. These findings collectively indicate that penfluridol exerts multifaceted antitumor effects by targeting key oncogenic pathways, providing experimental evidence for repurposing this antipsychotic agent as a potential therapeutic strategy against gastric cancer.