Esophageal cancer is one of the most difficult malignancies to cure, and identification of novel prognostic markers for patients with this disease is important. CD24 is a small highly glycosylated mucin-like protein. Several studies have shown that higher CD24 expression is significantly associated with shorter patient survival in various malignant tumors. However, the expression of CD24 and its clinicopathological significance in esophageal cancer remain largely unknown. Immunohistochemical analyses of CD24 and Ki-67 overexpression in 151 cases of esophageal squamous cell carcinoma were performed to examine the relationships of CD24 expression with clinicopathological parameters and patient survival. Five cell lines derived from esophageal cancer were subjected to Western blot analyses to evaluate CD24 expression. Immunohistochemically, CD24 expression was judged to be positive in 61 (40.4%) cases. CD24 expression was associated with lymph node metastasis (p = 0.005), pathologic stage (p = 0.018), number of nodal metastases (p = 0.003), lymphatic invasion (p = 0.002), venous invasion (p < 0.001), and Ki-67 labeling index (p < 0.001). The Pearson's correlation coefficient between the CD24 expression score and the Ki-67 labeling index was 0.404 (p < 0.001). CD24 expression was associated with disease-free survival (p < 0.001). A Cox multivariate regression analysis revealed that CD24 expression was an independent prognostic factor (p = 0.033). On Western blot analysis, CD24 was detected in all five cell lines. We conclude that overexpression of CD24, which was correlated with Ki-67 expression, is a novel independent prognostic marker for identifying patients with poor prognosis after curative resection of esophageal squamous cell carcinoma.