Liver cancer, one of the most common malignant tumors occurred worldwide, has emerged as a main health trouble and accounts for leading cancer-related death. Diosgenin is provided as an important material in the pharmaceutical industry, and is used to manage various medical troubles such as cancer because of its multiple bioactivities. DEAD box polypeptide 3 (DDX3) is involved in cancer biogenesis and modulates cancer progression. However, the role of DDX3 in human hepatocellular carcinoma (HCC) has not been fully understood. In the present study, we investigated the anti-tumor effects of diosgenin on HCC cells and whether DDX3 is involved in its antitumor activity. We observed that diosgenin dramatically inhibited cell proliferation, triggered apoptotic cell death, induced G2/M phase arrest, suppressed cell migration and invasion abilities. Moreover, the expression of DDX3 was measured and the results showed that DDX3 was significantly up-regulated upon diosgenin exposure. All together, our data indicated that diosgenin shows a cytotoxic effect on HCC cells and has potential therapeutic values for HCC patients.