Cyclooxygenase-2 (COX-2) is well established to play an important role in the tumorigenesis of a variety of human cancers; however, the function of COX-2 in the development of esophageal squamous cell carcinoma (ESCC) remains less clear. Here, we determined, first, the pattern of COX-2 expression in normal esophageal mucosa, dysplasia, carcinoma in situ (CIS) and invasive SCC. Immunohistochemical analysis showed that, while COX-2 was weakly expressed, if at all, in normal squamous epithelium, strong COX-2 expression was detected as early as the stage of dysplasia and frequently in 20 of 26 (77%) CIS and 86 of 111 (77%) invasive SCC. Upregulation of COX-2 in ESCC was found to be significantly associated with tumor progression (R = 0.493, P < 0.01). Further, treatment of human ESCC cell lines (KYSE450 and KYSE510) with NS-398, a COX-2 specific chemical inhibitor, suppressed the production of prostaglandin E2 (PGE2) and induced cell growth inhibition, cell cycle arrest at the G1-S checkpoint, and the expression of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. Finally, knockdown expression of COX-2 in KYSE450 cells by a specific COX-2 siRNA dramatically inhibited PGE2 production, cell growth and, more importantly, colony formation and tumorigenesis in nude mice. Together, this study suggested that COX-2 may be involved in an early stage of squamous cell carcinogenesis of the esophagus and has a non-redundant role in the regulation of cellular proliferation and tumorigenesis of esophageal epithelial cells.