AIMS: DLGAP5 plays a significant role in promoting cancer progression across various cancers. However, the specific role of DLGAP5 in neuroendocrine differentiation (NED) of prostate cancer (PCa) remains elusive. Our objective is to explore the mechanism by which DLGAP5 mediates NED in PCa. MATERIALS AND METHODS: Utilizing diverse public databases, we conducted bioinformatics analysis to examine DLGAP5 expression in PCa. We confirmed aberrant DLGAP5 expression in various PCa cell lines through Western blotting. Functional assays both in vivo and in vitro have validated the oncogenic role of DLGAP5 in PCa. Furthermore, we sought to identify downstream key genes to elucidate the mechanisms underlying DLGAP5-mediated NED in PCa. We also identified a small molecule drug, AAPK-25, which specifically targets DLGAP5. KEY FINDINGS: DLGAP5 was highly expressed in NEPC. The suppression of AR signaling promoted DLGAP5 transcription. DLGAP5 endowed PCa cells with a robust ability to proliferate and migrate. E2F1 was an important downstream target of DLGAP5. DLGAP5 mediated the NED of PCa through E2F1. AAPK-25, as an inhibitor of DLGAP5, inhibited PRAD proliferation by repressing the DLGAP5/E2F1 axis both in vitro and in vivo. SIGNIFICANCE: We identified the AR/DLGAP5/E2F1 signaling pathway as a pivotal mechanism that facilitates the transition of PCa towards a neuroendocrine phenotype. This pathway may represent a promising therapeutic target for NEPC patients.