ATP-binding cassette, subfamily G, member 2 (ABCG2) is a new member of the superfamily of ATP-binding cassette transporter proteins and known to be not only a member of the membrane transporters implicated in multidrug resistance, but also a molecular determinant of the side population phenotype, characteristics of which are reminiscent of stem cells. The aim of the current study was to clarify the significance of ABCG2 expression in esophageal squamous cell carcinoma (ESCC). We immunohistochemically investigated paraffin sections of 100 ESCC tumors and assessed the expression level of ABCG2 mRNA in 33 specimens by quantitative RT-PCR. In the immunohistochemical study, ABCG2 expression was detectable in 61% of patients and the proportion of ABCG2-positive cells was variable (0–100%). Interestingly, the presence of ABCG2-positive cells in the tumor, regardless of their amount, was associated with poorer survival (p= 0.0088). Moreover, it was revealed to be an independent prognostic factor along with the extent of the primary tumor and positive lymph node metastasis in multivariate analysis using Cox’s regression model. In the quantitative RT-PCR study, higher tumor ABCG2 mRNA expression was associated with poorer survival (p= 0.017). The absolute presence of ABCG2-positive cells in the tumor is a single independent prognostic factor, suggesting the underlying roles in malignant characteristics of ESCC other than drug resistance.