During early vertebrate embryogenesis, maternal Wnt/β-catenin signaling is thought to locally initiate expression of dorsal-specific genes. Here, eaf1 and eaf2 were identified as important maternal and zygotic modulators of Wnt signaling to initiate and specify ventral genes. Expression of ventral ved, vent, and vox was all obviously enhanced in either maternal or zygotic eaf1/2 morphants, and in both eaf1 heterozygous and homozygous mutants, but their expression was suppressed in embryos with over-expression of eaf1/2. Additionally, eaf1/2 were revealed to suppress ventral fates in embryos via Wnt/β-catenin1/Tcf signaling, complimentary to their roles in suppressing dorsal fates via Wnt/β-catenin2 signaling. Moreover, eaf1/2 were also revealed to obviously suppress the expression of axin2 induced by β-catenin2 rather than by β-catenin1, and the dorsal expression of axin2 in embryos was obviously suppressed by ectopic expression of eaf1/2. This study uncovers a novel dorsal-ventral patterning pathway, with eaf1 and eaf2 inhibiting ventral cells via suppressing Wnt/β-catenin1/Tcf signaling and inducing dorsal cells indirectly via suppressing β-catenin2-induced-axin2 on the dorsal side of embryos.