We previously reported embryonic expression pattern of strawberry notch (sbno) family genes, suggesting involvement in brain development. However function of sbno genes in the vertebrate development has not been known yet. Utilizing zebrafish embryos, we experimentally examined function of sbno genes during brain development in this report. Knockdown experiments of sbno1 and sbno2a disrupted brain morphology, and delayed developmental alteration of gene expression. The earliest effect of loss of function of sbno genes on the zebrafish embryogenesis that we found here was downregulation of otx2 expression. Knockdown of sbno1 specifically affects regionalization along the anterior-posterior axis of the brain. These results suggest essential roles of sbno genes in vertebrate brain development.