Down-regulation of GADD45β, which is known to influence cell growth control, apoptosis, and cellular response to DNA damage, has been verified to be specific in hepatocellular carcinoma and consistent with the degree of malignancy. Here, we identified promoter elements for several transcriptional factors in the proximal promoter of GADD45β using the luciferase assay. As a methyl donor for biological transmethylation reactions, S-adenosylmethionine (SAMe) could restore GADD45β expression in HepG2 in Northern blot analyses and quantitative real-time polymerase chain reaction. Activity and binding capacity of nuclear factor (NF)-κB were confirmed to be specifically induced by SAMe, as evidenced by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and a decrease of IκBα in Western blot analyses. The most upstream NF-κB-binding site was crucial for transcriptional activation. In contrast to NF-κB, although there is an E2F-1-binding site adjacent to the NF-κB sites, treatment with SAMe could not induce E2F-1-binding activity. Despite showing a similar GADD45β promoter regulatory pattern as HepG2 (p53 wild type), Hep3B (p53-null) did not exhibit GADD45β induction by SAMe, and the induction could be partially recovered on reconstituting p53 in Hep3B. Thus, our results suggest that GADD45β induction by SAMe via NF-κB may represent a novel mechanism of SAMe-mediated hepatoprotection, with p53 playing an important role.