Objective CXCL12/CXCR4 has been studied as an important biomarker in numerous human malignancies, but studies are limited in esophageal squamous cell carcinoma (ESCC). CXCL12/CXCR4 may be involved in the development of ESCC. In the present study, we evaluated the effects CXCL12/CXCR4 on the metastatic potential of tumor cells in vitro. Methods An effective RNAi sequence targeting the CXCR4 gene was selected, and a lentivirus shRNA vector was constructed to specifically silence CXCR4 expression in the ESCC cell line EC9706. Then, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blot were used to evaluate its effects on the metastatic potential of tumor cells in vitro. Results CXCR4 expression in EC9706 was significantly decreased after transfection with lentivirus shRNA vector. Downregulation of CXCR4 was accompanied with reduction of MMP-9. In addition, after silencing CXCR4 expression, the metastatic potential of EC9706 cells was inhibited considerably. Conclusion The downregulation of CXCR4 expression upon transfection with a lentivirus shRNA vector could inhibit the metastatic potential of ESCC cells. Our results may provide useful information regarding gene therapy for ESCC patients.