Among these changes, the activation of oncogenes and inactivation of tumor suppressor genes may be key steps for initiating tumor formation and development. We previously compared normal human nasopharynx mucosa and oral cavity mucosa of the soft palate using differential display and identified full-length NESG1 specifically expressed in human nasopharynx and trachea. In a subsequent investigation, we revised the open reading frame sequence of NESG1 and updated its version number from NM_012337.1 to NM_012337.2 in the NCBI GeneBank database. NESG1 was found to be specifically expressed in the nasopharynx epithelium and decreased or absent in NPC tissues and cell lines compared to normal tissue. In addition, the levels of NESG1 protein were significantly greater in the lower-grade NPC tissues versus highergrade NPC. Elevated HhAntag691 expression of NESG1 in NPC cells not only significantly decreased cell proliferation and G1-S phase transition, but also markedly inhibited cell migration, invasion, and in vivo tumorigenesis. NESG1 also significantly regulated the expression of cell cycle regulators CCNA1 and p21. Our findings provided evidence that NESG1 may act as a tumor suppressor in NPC. In this study, we present further evidence that NESG1 protein is downregulated in human NPC tissues and NPC cells compared to noncancerous nasopharynx tissues. We also show that reduced protein expression of NESG1 is inversely associated with NPC progression and poor prognosis. Downregulation of overexpressed NESG1 in 2F4 NPC cells significantly regained cell proliferation, migration, and invasion. Furthermore, NESG1 knockdown elevated CCNA1 expression and suppressed p21 expression. Gene expression profile analysis indicated that NESG1 participates in multiple pathways, such as MAPK signaling and tight junction formation regulation. Finally, an epigenetic evaluation of the NESG1 promoter revealed a lack of methylation, suggesting involvement of other mechanisms in NESG1 suppression during NPC. Our studies firstly demonstrate that NESG1 as a potential tumor suppressor is an unfavorable prognostic factor for NPC. Nasopharyngeal epithelium-specific gene NESG1 was initially isolated from human normal nasopharynx mucosa using an improved differential display approach, and its sequences was submitted to the Genbank database by our research group in 1999. In a recent investigation, we updated the NESG1 ORF sequence and studied its role in NPC cells. Our results preliminarily suggested NESG1 function as a tumor suppressor in NPC. In the present study, we presented additional support for this notion as NESG1 protein was downregulated in human NPC tissues and cells compared to noncancerous nasopharynx tissues by western blot. Furthermore, we also found that protein expression of NESG1 was progressively decreased in atypical hyperplasia and cancer tissues compared to normal and squamous epithelium by immunohistochemistry.