Pellino proteins are associated with immune and stress responses through their effects on NF-kappa B signaling and B-cell development, and through their role as a scaffold in TLR/IL-1R signaling pathways. However, their function during embryonic development is unclear. Here, we report the developmental expression patterns and functions of pelt1b, which encodes a zebrafish ortholog of human Pellino1. Maternal peli1b transcripts were present in zebrafish embryos at the 1-cell stage and zygotic transcripts appeared in the shield area at 6 hours post fertilization (hpf), particularly in the neural plate of the dorsal region. peli1b transcripts were concentrated in the somites, lens, myogenic cells, lateral plate mesoderm, and presomitic mesoderm at 12 hpf, but expression shifted to the telencephalon, diencephalon, hindbrain, and rhombomeres (r1-7) at 24 hpf. Distribution of peli1b transcripts was further restricted to the telencephalon, diencephalon, hindbrain, eyes, and pectoral fins at 48 hpf. Knock-down of peli1b with a peli1b antisense morpholino resulted in significant developmental defects and a reduction in size of the telencephalon, diencephalon, rhombomeres (r1-7), and spinal cord at 24 hpf. When peli1b-knock-down embryos were analyzed for zic3, a marker associated with the central nervous system, we found lower levels of zic3 transcripts in the shield area at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, and hindbrain at 14 hpf. Finally, the ERK3/4 inhibitor SB203580 also induced a significant reduction in the level of zic3 transcripts in the neural plate at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, hindbrain, segmental plate, dorsal spinal cord, and dorsal posterior neural plate at 14 hpf. It is thus likely that the association between Peli1b and brain development in zebrafish embryos occurs via ERK3/4 pathways.