The chemokine CXCL12, also known as stromal cell-derived factor-1 and its receptor CXCR4 have been shown to play prominent roles in regulating the directional migration and proliferation of various types of cancer cells during the metastatic process. However, few researchers have examined the expression of CXCL12 and CXCR4 and their prognostic value in patients with esophageal squamous cell carcinoma (ESCC). We investigated immunohistochemically the relationship between CXCL12 and CXCR4 expression and clinicopathological factors including prognosis in surgical specimens of primary tumors in 214 patients with ESCC. The positive expression rate of CXCL12 was 53.7% and that of CXCR4 was 84.6%. Positive CXCL12 expression was significantly correlated with lymph node metastasis, tumor stage, gender and lymphatic invasion. The overall and disease-free survival rate was significantly lower in patients with positive CXCL12 expression than in those with negative CXCL12 expression. The expression of CXCR4 had no correlation with clinicopathological variables and prognosis. We showed that positive CXCL12 expression was related to a greater degree to tumor development, compared with CXCR4 expression. Evaluation of CXCL12 expression is useful for determining tumor properties, including nodal metastasis and prognosis in patients with ESCC.