Background: p53 deletion and mutation as well as upregulation of alpha-fetoprotein (AFP) are hallmarks of hepatocarcinogenesis. p63 and p73 belong to the family of p53-related transcription factors expressing a variety of isoforms. The expression of dominant negative (Delta N) p73 is related to the reduced survival of patients with hepatocellular carcinoma (HCC). In this study, we characterized the interaction between p53 family-dependent signaling pathways and the regulation of AFP at the gene and protein levels as essential determinants of therapeutic response and prognosis in HCC. Methods: Putative p53-, p63-and p73-binding sites within the AFP gene were identified in silico. Hep3B cells were transfected with plasmids encoding for p53, p63 and p73 to analyze the interplay of the p53 family with AFP. AFP transcription was determined by RT-qPCR. Protein levels of AFP, p53, p63 and p73 were analyzed by Western blot. Results: Underlining the importance of the crosstalk between the p53 family-dependent pathways and AFP regulation we identified eight novel putative binding sites for the members of the p53 family within the introns 1, 2, 3, 4, 7, 8, 11, and 12 of the AFP gene. Accordingly, full-length isoforms of p53, p63 and p73 efficiently downregulated AFP both on mRNA and protein level. Thus, the p53 family members were identified to be major regulators of AFP repression. Of note, p63 was characterized as a novel and p73 as the most efficient repressor of AFP. Conclusion: p53 mutation and upregulation of AFP are essential oncogenic events in the development of HCC. Here we show that AFP gene regulation occurs via a combined action of the p53 family members p53, p63 and p73. All three tumor suppressors reduce AFP gene and protein expression. Thus, our findings reveal a novel interaction of p53 family-dependent signaling pathways and AFP regulation at the gene and protein levels in HCC.