hHSS (human hepatic stimulator substance), acting as a hepatotrophic growth factor, promotes liver regeneration. However, the regulatory mechanisms for hHSS transcription are still poorly understood. In the present study, we investigated transcription of hHSS triggered by EGF (epidermal growth factor) and the role of C/EBPβ (CCAAT/enhancer-binding protein β) as a potential core factor responsible for hHSS transcription in HepG2 cells. The results show that EGF suppresses hHSS mRNA expression at early time points. Using a promoter deletion assay, we identified a proximal region (-358/-212) that is required for EGF suppression. Overexpression of C/EBPβ enhances EGF suppression of hHSS, and mutation of the C/EBPβ-binding site at -292/-279 or siRNA (short interfering RNA) interference abolishes EGF suppression. Furthermore, using an electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we found that C/EBPβ specifically binds to the -292/-279 site that is responsible for EGF inhibition. Moreover, using a knockin (overexpression) and knockdown strategy (siRNA), we confirmed that C/EBPβ is a key factor responsible for inhibition of hHSS mRNA expression. Pre-treatment with an inhibitor of JNK (c-Jun N-terminal kinase) or down-regulation of JNK1 with specific siRNA reverses EGF-inhibited hHSS expression. Our results provide a crucial regulatory mechanism for EGF in hHSS transcription within the promoter proximal region.