Oxidored-nitro domain containing protein 1 (NOR1) is a novel member of the nitroreductase family that was first isolated as a tumor suppressor gene from human nasopharyngeal carcinoma (NPC). However, the role of NOR1 gene dysfunction in human cancers has not been addressed. We analyzed the expression of NOR1 in various human cancer and benign tissue specimens and found significant downregulation in nine types of cancer compared with corresponding non-tumor tissues. The recombinant expression vector pCDNA3.1-myc-his-NOR1 was constructed and transfected into human NPC 6-10B nasopharyngeal cancer and HeLa cervical cancer (CCA) cells. We found that stable NOR1 overexpression resulted in suppression of 6-10B and HeLa cell proliferation and led to S phase cell cycle arrest. In addition, NOR1 upregulation enhanced apoptosis in pCDNA3.1-myc-his-NOR1 stably transfected cells, and it also altered the expression of proteins involved in the mitochondria-dependent apoptotic pathway. Furthermore, we also found that the NOR1 protein is a cytoplasmic protein that is partially localized in the mitochondria and endoplasmic reticulum. Therefore, NOR1 is an important tumor suppressor gene associated with NPC and CCA and may play antitumor roles by inhibiting proliferation, preventing colony formation, and promoting the apoptosis of tumor cells via the mitochondrial-dependent apoptotic pathway. However, the precise mechanism behind the NOR1 antitumor effects needs to be investigated further.