It is well established that the gbx2 homeobox gene contributes to the positioning of the midbrain-hindbrain boundary (MHB) governing the development of adjacent brain regions in vertebrate embryos, but the specific aspects of the gene regulatory network regulated by gbx2 during brain development remain unclear. In the present study, we sought to comprehensively identify gbx2 target genes in zebrafish embryos by microarray analysis around the end of gastrulation, when the MHB is established, using transgenic embryos harboring heat-inducible gbx2. This analysis revealed that a large number of genes were either upregulated or downregulated following gbx2 induction, and the time course of induction differed depending on the genes. The differences in response to gbx2 were found by functional annotation analysis to be related to the functions and structures of the target genes. Among the significantly downregulated genes was her5, whose expression in the midbrain was precisely complementary to gbx2 expression around the MHB, suggesting that gbx2 expression in the anterior hindbrain restricts her5 expression to the midbrain. Because her5 represses neurogenesis, gbx2 may positively regulate neural development in its expression domain. Indeed, we showed further that gbx2 induction upregulated neural marker expression in the midbrain. Quantitative PCR analysis revealed that gbx2 upregulated the expression of the zebrafish proneural gene ebf2, whereas it repressed notch1a, which generally represses neurogenesis. Taken together, these results demonstrate that gbx2 not only functions to position the MHB but also regulates neurogenesis in the anterior hindbrain.