Our previous results revealed that FAM172A was significantly downregulated in liver tissue from hepatocellular carcinoma or cirrhotic patients. The present study was designed to elucidate the regulatory role of FAM172A in HepG2 cells. In order to determine the expression of the FAM172A protein, western blot analysis was performed. Confocal laser scanning technique was used to observe the localization of FAM172A in HepG2 cells. Surface plasmon resonance experiments were used to determine the binding activity of FAM172A and active single sugar and Ca2+. The cell cycle progression of HepG2 cells was assessed by flow cytometry. The FAM172A protein was localized in the endoplasmic reticulum of HepG2 cells. This protein was moderately expressed in normal liver tissue, but was significantly decreased in liver tissue of patients with chronic hepatitis B When co-cultured with the FAM172A recombinant protein, HepG2 cells exhibited complete cell cycle arrest in the S phase at a high concentration (100 ng/ml). Proliferation of HepG2 cells treated with the FAM172A recombinant protein was prominently inhibited compared with that of the control cells. Western blot analysis showed that upregulation of Notch 3 and cyclin E may be related with the cell cycle control. Our results indicate that FAM172A may be a novel tumor-suppressor gene, which plays an important role in cell cycle control and tumor cell proliferation. G1/S phase arrest may be mediated, at least partially, by the Notch 3 signaling pathway.