Eaf family genes act in multiple cellular responses such as tumor suppression and embryonic development. In our previous work, Eaf1/2 was found to modulate convergence and extension (C&E) movements and pattern the embryonic anterior-posterior axis during zebrafish embryogenesis. Here, we found that loss-of-function of eaf1/2 caused expanded mesoderm and endoderm in zebrafish embryos and led to the recovery of endoderm specification in TGF-β factor-mzoeptz257 mutants, while gain-of-function of eaf1/2 induced reduced mesoderm and endoderm. Analyses of gene expression profiles in Eaf deleted or over-expressed mammalian cells indicated that the roles of Eaf1 and Eaf2 in inhibiting TGF-β signals were conserved from fish to mammals. By taking advantages of TGF-β reporters, eaf1/2-fused engrailed proteins, and P53M214K mutant, we revealed that Eaf1 and Eaf2 might suppress TGF-β transduction by synergistically inhibiting none-P53 and P53-required TGF-β signaling. Furthermore, Eaf1/2 might co-localize and interact with TGF-β transcriptional factors in the transcriptional complex as repressors to target and suppress TGF-β signaling activity. Our study unveiled a previously unrecognized link of Eaf1/2 genes with TGF-β and P53 in vertebrates and demonstrated a conservation of TGF-β suppression activity for Eaf1/2 family genes from fish to mammals, which might shed some light on the molecular mechanistic basis of Eaf1 and Eaf2 in tumor suppression.