Sox superfamily proteins are DNA-binding transcriptional factors that contain highly conserved high-mobility group (HMG) box and take part in various development process. Sox31 is a maternal factor supplied in the oocyte and starts its zygotic expression during mid-blastula transition (MBT). From gastrulation stage, it mainly resides in neural tissue. Ectopically expression of Sox31 mRNA leads to cyclopia, fusion eyes, or totally loss of anterior head structure, in accompany with severe notochord defects. Molecular markers indicate that forebrain tissue reduces sharply while the posterior neural tissue expands anteriorly. In addition, organizer specification is also suppressed. Oppositely, an antisense morpholino designed functionally knockdown Sox31 causes typically dorsalized phenotype and reversed central nervous system (CNS) anteroposterior (AP) patterning. Gain of function with chimeric construct, where Sox31 HMG DNA binding domain is fused to a transcription activation domain (VP16) or transcription suppression domain (EnR), suggests that Sox31 acts as a transcriptional suppressor in vivo. The expression of Bozozok (Dharma), a direct target gene of pre-MBT Wnt/β-catenin signal, is suppressed by Sox31. Thus, to unveil the relationship between Sox31 and β-catenin-related transcriptional activity, we designed Top/Fop luciferase assay in HEK293T cells, and found that Sox31 could indeed suppress Tcf/Lef-dependent transcriptional activity without influencing the stability of β-catenin. Moreover, post-MBT Wnt signal was reduced in Sox31 morphants corresponding to the suppressed hindbrain structure, while phenotypic defects caused by excessive Sox31 could be rescued by Wnt antagonist dkk1. Taken together, Sox31 functions as an essential CNS AP patterning determinant and coordinates the CNS AP patterning process with organizer specification.