AbstractBackground and Aim:  Cyclooxygenase‐2 (COX‐2) plays an important role in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). However, it is not clear whether COX‐2 is involved in the early or late stage of the development of ESCC. The aim of this study was to investigate the role of COX‐2 in the carcinogenesis of ESCC by an immortalized esophageal epithelial cell line.Methods:  Human papillomavirus type 16 (HPV16)–E6/E7 and human telomerase reverse transcriptase (hTERT) transfection were used for immortalization of esophageal epithelial cells. COX‐2‐specific RNA interference was used for the inhibition of COX‐2 expression.Results:  An immortalized esophageal epithelial cell line, NE6‐E6E7/hTERT, was established, which had high proliferation activity but failed to induce colony formation in soft agar. COX‐2 expression was upregulated in the early process of immortalization, while COX‐2 small interfering RNA (siRNA) decreased the Bcl‐2 expression, increased the expression of Bax, and induced cell‐cycle arrest at the G0/G1 phase in NE6‐E6E7/hTERT cells. Expressions of p53, cyclinD1, and the ratio of hyperphosphorylated‐RB/hypophosphorylated‐RB were progressively increased after E6E7 and the subsequent hTERT transfections. These changes were accompanied by the alteration of COX‐2 expression, but could be reversed by COX‐2 siRNA (P < 0.05). P16 expression was significantly downregulated in NE6‐E6E7 or NE6‐E6E7/hTERT cells (P < 0.05), and was not affected by COX‐2 siRNA.Conclusions:  Our results suggest that induction of cyclooxygenase‐2 is essential in the human papillomavirus type 16 and hTERT‐induced immortalization of human esophageal epithelial cells, and that COX‐2 inhibition may be a potential target to block the carcinogenesis of ESCC at the precancerous stage.