Trametes robiniophila Murr. (Huaier) is a widely used anti-cancer agent in China. Strong evidence for the anti-proliferative activity of Huaier has been reported; however, its anti-metastatic potential against gastric cancer (GC) as well as its underlying mechanism of action are unknown. Here, we show that treatment with an aqueous Huaier extract over a range of concentrations significantly suppressed both the invasiveness and migratory ability of GC cells. Huaier could also partly reverse the epithelial-mesenchymal transition (EMT), as characterized by increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal markers N-cadherin and vimentin. In addition, Huaier-treated cells expressed lower levels of Twist compared to untreated controls, and overexpression of Twist via transfection could partially abolish the anti-metastatic activity of Huaier. Furthermore, elevated Twist expression was correlated with an advanced TNM stage, a high rate of lymph node metastasis, and reduced disease-free survival in GC patients. These findings reveal a novel anti-metastatic mechanism for Huaier, which inhibits the EMT by targeting Twist, suggesting its potential application against a GC relapse.