• MTE inhibits the proliferation of PCa cells by inducing apoptosis • MTE reduces Atg5/Atg12 protein expression and promotes nuclear translocation of FoxO3a • MTE-induced apoptosis relies on the inhibition of autophagy and FoxO3a activation in PCa cells Prostate cancer (PCa) is one of the most commonly diagnosed malignant tumors in older men, causing high morbidity and mortality along with significant economic and medical costs. Marsdenia tenacissima extract (MTE) has been approved for the treatments of esophageal, gastric, lung, and liver cancers in China. Recently, it has been reported that MTE exhibits a promising therapeutic effect on PCa, but the underlying mechanism is unclear. To investigate the action mechanisms of MTE against PCa. HPLC-CAD-QTOF-MS/MS technology was performed to analyze the component characterization of MTE. Effects of MTE on PCa cell proliferation, apoptosis, and autophagy were assessed using CCK-8 assays, Hoechst staining, TUNEL staining, transmission electron microscopy, and acridine orange staining. The key molecules and important signaling pathways regulated by MTE in PCa cells were explored using proteomics and qRT-PCR technology. FoxO3a expression and localization were monitored by western blot, immunofluorescence, and immunohistochemistry assays. Rapamycin, an inducer of autophagy, was used to investigate the relationship between MTE-induced autophagy and apoptosis. The PC3 xenograft mouse model was employed to verify the anti-PCa effect and underlying mechanism of MTE in vivo . In total, HPLC-CAD-QTOF-MS/MS analysis identified 21 components of MTE. MTE significantly inhibited the cell proliferation of several PCa cell lines. In PC3 and C4-2 cells, MTE treatment significantly induced apoptosis, increased cleaved PARP and Bax expressions, and decreased Bcl-2 expression. Proteomic and qRT-PCR analyses revealed that the anti-PCa activity of MTE was closely related to the autophagic degradation of FoxO3a. Moreover, MTE significantly inhibited autophagy via downregulating the expression of Atg5/Atg12. More importantly, MTE significantly increased the expression and nuclear translocation of FoxO3a. Interestingly, Rapamycin reversed MTE-induced apoptosis and FoxO3a protein accumulation. Finally, in PC3 xenograft mice, MTE treatments significantly suppressed tumor growth, induced tumor cell apoptosis, inhibited tumor cell autophagy, and activated FoxO3a nuclear translocation. The present study demonstrated that MTE inhibits the autophagic degradation of FoxO3a, which promotes FoxO3a nuclear translocation and ultimately induces PCa apoptosis. Our data provide new insights into the role of MTE as a potential treatment for PCa.