Zebrafish pou5f1, also known as pou2, encodes a POU-family transcription factor that is transiently expressed in the prospective midbrain and anterior hindbrain during gastrulation, governing brain development. In the present study, we found that the main regulatory elements reside in the proximal upstream DNA sequence from -2.2 to -0.12 kb (the -2.2/-0.1 region). The electrophoretic gel mobility shift assay (EMSA) revealed four functional octamer sequences that can associate with zebrafish Pou2/Pou5f1. The expression of mutated reporter constructs, as well as EMSA, suggested that these four octamer sequences operate in a cooperative manner to drive expression in the mid/hindbrain. We also identified a retinoic acid (RA)-responsive element in this proximal region, which was required to repress transcription in the posterior part of the embryo. These data provide a scheme wherein pou2/pou5f1 expression in zebrafish embryos is regulated by both an autoregulatory loop and repression by RA emanating from the posterior mesoderm.