Dopamine receptor agonist (DA)-resistant prolactinoma presents a significant clinical challenge, highlighting the need for novel therapeutic strategies. Deguelin is a rotenoid compound derived from several plant species with unique antitumor effects. In this study we investigated the efficacy of deguelin on DA-resistant prolactinoma and elucidated its antitumor mechanisms. We showed that deguelin concentration-dependently inhibited cell viability, proliferation and prolactin secretion, and promoted apoptosis and cell cycle arrest in two prolactinoma tumor cell lines GH3 and MMQ. In CCK-8 assay, the IC50 values of deguelin for GH3 and MMQ were 0.1518 and 0.2381 mu M, respectively. Network pharmacology analysis predicted that ornithine decarboxylase (ODC), a rate-limiting enzyme in the de novo synthesis of polyamine and responsible for converting ornithine into putrescine, was the target of deguelin. We demonstrated that deguelin directly interacted with ODC, competitively inhibiting putrescine production, and thereby reducing the levels of active Rac1. Transcriptomic analysis of deguelin-treated GH3 cells identified the PI3K/AKT signaling pathway as a crucial mediator of the action of deguelin with the inhibition of CREB3L1 playing a central role. In GH3 xenograft nude mice, administration of deguelin (4 mg/kg, i.p., every other day for two weeks) significantly inhibited tumor growth with significant reduction in both AKT phosphorylation and CREB3L1 levels in deguelin-treated xenografts. These results suggest that deguelin can be considered a therapeutic option for treating DA-resistant prolactinoma and serve as a basis for developing novel treatment approaches.