Gankyrin, a recently discovered oncoprotein, is a promising target for drug therapy because it is overexpressed in most hepatocellular carcinomas. Since gankyrin interacts with MAGE-A4, we made several MAGE-A4 mutants and assessed their effects on cell growth. We found that the C-terminal 107 amino acids of MAGE-A4 (MAGE-A4DeltaN1) induced p53-dependent and p53-independent apoptosis. MAGE-A4DeltaN1 increased the p53 protein level, but decreased the p21(Cip1) transcript and protein levels. During apoptosis Bcl-xL was down-regulated and mitochondrial integrity was disrupted. A yeast two-hybrid screen identified Miz-1 as a MAGE-A4DeltaN1-binding protein. MAGE-A4DeltaN1 was recruited through association with Miz-1 to the p21(Cip1) promoter and down-regulated transcription of p21(Cip1). In 293T cells and U-2 OS cells, full-length MAGE-A4 was processed to generate a C-terminal fragment of 104 amino acids with activities similar to MAGE-A4DeltaN1. Processing was inhibited with a broad range caspase inhibitor Z-VAD-FMK, but not by site-directed mutagenesis of aspartic acids in MAGE-A4, suggesting an indirect involvement of caspase(s) in the processing. The amount of the processed form was increased by exposure of cells to adriamycin. Transduction with a HIV Tat-MAGE-A4DeltaN1 fusion protein suppressed anchorage-independent growth of gankyrin-overexpressing cells in vitro and in vivo. These results demonstrate that the C-terminal fragment of MAGE-A4 induces apoptosis at least partly by binding to Miz-1, and that the fragment may be exploited as an anticancer agent. Furthermore, the finding that a C-terminal fragment with pro-apoptotic activity is generated from full-length MAGE-A4 after genotoxic stress in human cells suggests a novel function for MAGE-A4.