Esophageal cancer is one of the most common cancer types in the world, with a widely varying incidence between different regions. Zinc deficiency (ZD) is very common in high-risk areas for esophageal cancer. Dietary ZD is reported to be associated with esophageal squamous cell carcinoma (ESCC). In the current study, the effects of ZD on tumorigenesis and expression of inflammatory factors were investigated in mice. It was identified that a ZD diet advanced ESCC and increased the expression of cyclooxygenase-2 (COX-2) prior to the occurrence of ESCC in mice. ZD significantly enhanced DNA methyltransferase (DNMT) activity and increased the expression of DNMT1 and DNMT3B. Furthermore, the expression of miR-128 was downregulated by methylation, and COX-2, a direct target of miR-128, was upregulated with the reduction in miR-128. Upregulation of miR-128 inhibited the cell cycle, proliferation and metastasis, and the expression of COX-2, cyclin D1 and retinoblastoma protein (Rb). Furthermore, the relative expression level of miR-128 was negatively associated with COX-2 in ESCC tissues. Collectively, these findings indicate that methylation-associated silencing of miR-128 promotes the development of esophageal cancer through upregulation of the expression of cyclin D1 and Rb by targeting COX-2 in ZD regions with a high incidence of esophageal cancer.