Epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P< 0.05). Depletion of Twist by stable shRNA-mediated knockdown decreased the migratory ability of cancer cell lines in vitro. Suppression or overexpression of Twist also resulted in an altered expression of the molecular mediators of EMT. Furthermore, exogenous TGF-β promoted EMT by upregulating the expression of Twist through the TGF-β/Smad3 pathway, and this effect was eliminated by Twist depletion in cancer cells as demonstrated in the in vitro study. The use of in vivo models revealed a decreased tumor proliferation potential in Twist-depleted cancer cells. The results suggested a novel function for Twist in the promotion of EMT via TGF-β/Smad3 signaling pathway. Thus, Twist constitutes a potential therapeutic target in human cervical cancer.