Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, requiring effective biomarkers for prognosis and therapeutic responsiveness. In this retrospective study of banked pathology material, we investigated the protein expression of MMP-21 in ESCC and its association with clinical significance. MMP-21 protein expression was investigated in 311 cases of ESCC by immunohistochemistry assay. Statistical analysis was utilized to evaluate the association of MMP-21 expression with clinicopathological characteristics and overall survival of patients with ESCC. Results showed that MMP-21 expression was significantly increased in ESCC (P < 0.001). It was also found that MMP-21 expression in ESCC was associated with tumor invasion (P < 0.001), lymph node metastasis (P < 0.001), distant metastasis (P < 0.001) and TNM stage (P < 0.001). Kaplan-Meier analysis showed MMP-21 expression was associated with overall survival of patients with ESCC for patients with tumors of positive MMP-21 staining tend to have worse overall survival (P < 0.001). Multivariate analysis proved that MMP-21 was an independent prognostic factor for overall survival for patients with ESCC (P < 0.001). These results suggested the potential role of MMP-21 in tumor progression and prognosis predication of human ESCC. It might also be a novel molecular target for therapeutic intervention.