Hypoxia and oxidative stress are critical factors in carcinogenesis and exist throughout cancer development; however, the underlying mechanisms are far from clear. Here, for the first time to our knowledge, we reported that neuroglobin (Ngb), an intracellular hexa-coordinated globin serving as an oxygen/reactive oxygen species (ROS) sensor, functions as a tumor suppressor in hepatocelluar carcinoma (HCC). Ngb protein and mRNA expression were significantly down-regulated in tumor tissues, compared with its adjacent non-tumor tissues of human HCC samples and normal liver tissues. Knock-down of Ngb by RNA interference promoted human HCC cell line (HepG2) growth and proliferation, G0/G1-S transition in vitro, and tumor growth in vivo. On the contrary, overexpression of Ngb suppressed HepG2 cell growth and proliferation, G0/G1-S transition, colony formation in vitro, and tumorigenicity in vivo. These results established a tumor suppressor function of Ngb in HCC. The underlying mechanisms were further investigated. Overexpression of Ngb suppressed Raf/MEK/extracellular signal-regulated kinase (Erk), whereas knockdown of Ngb enhanced Raf/MEK/Erk activation in HepG2 cells in vitro and in vivo. Glutathione S-transferase pull-down showed that Ngb interacted with c-Raf-1 in HepG2 cells. Overexpression of Ngb suppressed serum- and H₂O₂-stimulated Erk activation in HepG2 cells. Pharmacological inhibition of Erk activation abolished the proliferative effect of Ngb knockdown in HepG2 cells. Mutation of Ngb at its oxygen-binding site (H64L) abolished the inhibitory effects of Ngb on Erk activation and HepG2 cell proliferation. Therefore, we propose that Ngb controls HCC development by linking oxygen/ROS signals to oncogenic Raf/mitogen-activated protein kinase (MAPK)/Erk signaling. Our data suggest that neuroglobin could be a new target for cancer therapy.