Prechordal mesendoderm (PME) is a derivative of gastrula organizer underlying the anterior neural plate of vertebrate embryos. It has been firmly established that PME is critical for head induction and anterior-posterior patterning. Therefore, the establishment of PME in a desired shape and size at a correct position during early embryogenesis is crucial for normal head patterning. However, it remains largely unclear how the desired form and size of PME is generated at a predestined position during early embryogenesis. Here we show that in zebrafish a maternal transcription repressor Vsx1 is essential for this early developmental regulation. Knocking down maternal vsx1 resulted in impaired PME formation and progression associated with a deficient and posteriorized forebrain. Loss- and gain-of-function experiments showed that maternal Vsx1 is essential for repressing ntl ectopic expression in more animal region at early gastrula stages. Chromatin immunoprecipitation assay in combination with core consensus sequence mutation analysis further revealed that maternal Vsx1 can directly repress ntl transcription by binding to the proximal promoter at a specific site. Simultaneous inhibition of ntl function could successfully suppress the defects of both PME and forebrain formation in maternal Vsx1 knockdown embryos. Our results reveal a pivotal role for maternal Vsx1 as a direct transcriptional repressor of ntl expression at the margin of the zebrafish gastrula to ensure directional cell polarization and migration of PME cells.