ErbB3 binding protein 1 (EBP1) has been recently reported to function as a tumor suppressor in the progression of multiple cancers, including breast cancer, prostate cancer, salivary adenoid cystic carcinoma (ACC), and oral squamous cell carcinoma (OSCC). However, the expression and physiological significance of EBP1 in hepatocellular carcinoma (HCC) remain unclear. In the study, we showed that EBP1 was significantly downregulated in clinical HCC specimens, and that decreased expression of EBP1 was associated with enhanced proliferation in HCC cells. Western blot and immunohistochemical analyses revealed that EBP1 was remarkably downregulated in HCC tissues compared with the adjacent normal ones. The levels of EBP1 were significantly associated with histological grade (P = 0.034), tumor size (P = 0.001), and Ki67 expression (P < 0.001) in HCC specimens. Univariate and multivariate analyses showed that EBP1 could serve as an independent prognostic indicator of patients' survival. Serum starvation and refeeding assay indicated that EBP1 was accumulated in growth-arrested HCC cells, and was progressively decreased when cells entered into S phase. Moreover, the depletion of EBP1 induced growth acceleration and cell cycle progression in L02 hepatocytes. On the basis of these findings, we conclude that EBP1 may be a valuable prognostic marker and promising therapeutic target of HCC.