Cervical cancer with early metastasis of the primary tumor is associated with poor prognosis and poor therapeutic outcomes. Since epithelial-to-mesenchymal transition (EMT) plays a role in acquisition of the ability to invade the pelvic lymph nodes and surrounding tissue, it is important to clarify the molecular mechanism underlying EMT in cervical cancer. RhoE, also known as Rnd3, is a member of the Rnd subfamily of Rho GTPases. While previous reports have suggested that RhoE may act as either a positive or a negative regulator of cancer metastasis and EMT, the role of RhoE during EMT in cervical cancer cells remains unclear. The present study revealed that RhoE expression was upregulated during transforming growth factor-β (TGF-β)-mediated EMT in human cervical cancer HeLa cells. Furthermore, reduced RhoE expression enhanced TGF-β-mediated EMT and migration of HeLa cells. In addition, we demonstrated that RhoE knockdown elevated RhoA activity and a ROCK inhibitor partially suppressed the acceleration of TGF-β-mediated EMT by RhoE knockdown. These results indicate that RhoE suppresses TGF-β-mediated EMT, partially via RhoA/ROCK signaling in cervical cancer HeLa cells.