Promoter hypermethylation-mediated silencing of tumor suppressor genes (TSGs) is a hallmark of oncogenesis. Oxidored-nitro domain-containing protein 1 (NOR1) is a candidate TSG that is downregulated in nasopharyngeal carcinoma (NPC). In the present study, we identified a functional NOR1 promoter that is regulated by heat shock factor 1 and nuclear respiratory factor 1. The promoter is located within a CpG island. Hypermethylation of this CpG island was found in NPC tissue samples and cancer cell lines, whereas no aberrant promoter methylation was detected in non-cancerous nasopharyngeal tissue samples or normal nasopharyngeal epithelial cells. Treatment of NPC 6-10B cells and leukemia HL60 cells with 5'-aza-2'-deoxycytidine increased endogenous levels of NOR1 messenger RNA. Ectopic expression of NOR1 in NPC HNE1 cells inhibited tumor cell colony formation and viability. These findings suggest that promoter hypermethylation may participate in transcriptional inactivation of the NOR1 gene in NPC. Frequent epigenetic inactivation of the NOR1 gene in NPC suggests that it may be a critical tumor suppressor involved in the development of NPC.