Mad protein has been shown as an antagonist of c-Myc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally. An eukarryotic vector pCDNA III containing full ORF fragment of mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned. Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the mad-transfected cells were partially inhibited in comparison to control cells. Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1 to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume. Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.