Objective To investigate the expression of SALL4, Bmi-1 and β-catenin in esophageal squamous cell carcinoma (ESCC) and the related clinical implications. Methods Immunohistochemical staining was used to detect the expression of SALL4, Bmi-1 and β-catenin in 70 normal esophageal mucosa specimens,70 dysplasia mucosa specimens and 123 ESCC specimens; and the relationship of their expression with the clinicopathological characteristics of ESCC was analyzed. Results The positive rates of SALL4, Bmi-1 and the aberrant rate of β-catenin expression gradually increased in order in normal esophageal mucosa, dysplasia mucosa and ESCC groups. The positive rates of SALL4, Bmi-1 and the aberrant rate of β-catenin expression in the dysplasia mucosa and ESCC groups were significantly higher than those in normal esophageal mucosa group (P<0.01); those in ESCC group was significantly higher than those in the dysplasia mucosa (P<0.01); and the positive rates of SALL4 were not significantly different between the dysplasia mucosa and ESCC groups (P>0.05). In the dysplasia mucosa group, the positive rate of Bmi-1 increased along with the degree of dysplasia (P<0.01). In the ESCC cases, the positive rate of Bmi-1 and aberrant rate of β-catenin were corelated with depth of invasion, degree of differentiation and lymph node metastasis of ESCC (P<0.05), and positive rate of SALL4 was correlated with the clinical staging (P<0.05) and lymph node metastasis of ESCC (P<0.01). The expression of SALL4, Bmi-1 and β-catenin in the 123 cases of ESCC were positively correlated with each other (SALL4 and Bmi-1: r=0.373,P<0.01; SALL4 and β-catenin: r=0.214, P<0.05; Bmi-1 and β-catenin: r=0.204, P<0.05). Conclusion SALL4, Bmi-1 and β-catenin might be involved in the development, progression, invasion and metastasis of ESCC; and the three of them might interact through corresponding signal pathways.