Oldhamianoside II is a novel triterpenoidsaponin that can be isolated from the roots of Gypsophila oldhamiana. In vitro and in vivo experiments have revealed that it inhibits tumor growth and metastasis in various types of tumor; however, the exact mechanism remains to be fully elucidated. In the present study, oldhamianoside II treatment in prostate cancer cells exerted substantial anticancer activity, including decreased cell proliferation and invasion. Mechanistically, oldhamianoside II was found to reverse the epithelial-mesenchymal transition (EMT), as demonstrated by its induction of E-cadherin and suppression of vimentin and N-cadherin at the mRNA and protein levels. Furthermore, oldhamianoside II treatment upregulated Wnt antagonist expression and promoted the proteasome-mediated degradation of β-catenin to inhibit the activity of β-catenin signaling. In summary, the present study revealed that oldhamianoside II exerts its antitumor effects via the regulation of EMT and β-catenin function, and further supports its potential for use in clinical treatment.