This study aimed to analyze the expression, clinical significance of B cell translocation gene 1 (BTG1) in hepatocellular carcinoma, and the biological effect in its cell line by BTG1 overexpression. Immunohistochemistry and Western blot were used to analyze BTG1 protein expression in 70 cases of hepatocellular cancer and 32 cases of normal tissues to study the relationship between BTG1 expression and clinical factors. Recombinant lentiviral vector was constructed to overexpress BTG1 and then infect hepatocellular cancer HepG2 cell line. The level of BTG1 protein expression was found to be significantly lower in hepatocellular cancer tissue than normal tissues (P< 0.05). Decreased expression of BTG1 was significantly correlated with tumor invasion, lymph node metastasis, clinic stage, and histological grade of patients with hepatocellular cancer (P< 0.05). Meanwhile, loss of BTG1 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P< 0.05). The result of biological function has shown that HepG2 cell-transfected BTG1 had a lower survival fraction; higher percentage of the G0/G1 phases; higher cell apoptosis; significant decrease in migration and invasion; and lower Cyclin D1 (CND1), B cell lymphoma 2 (Bcl-2), and matrix metalloproteinases (MMP)-9 protein expression compared with HepG2 cell-untransfected BTG1 (P< 0.05). BTG1 expression decreased in hepatocellular cancer and correlated significantly with lymph node metastasis, clinic stage, histological grade, poor overall survival, proliferation, and metastasis in hepatocellular cancer cell by regulating CND1, Bcl-2, and MMP-9 protein expression, suggesting that BTG1 may play important roles as a negative regulator to hepatocellular cancer cell.