Coloboma is a congenital ocular malformation caused by the failure of the choroid fissure to close during development. Although mutations in a number of genes have been identified, the cellular and molecular mechanisms in the pathogenesis of the disease remain poorly understood. Here we identify the secreted guidance molecule, Semaphorin3f (Sema3f), as a novel factor involved in retinal development. We used embryonic transgenic (Tg) zebrafish injected with antisense morpholino or CRISPR interference RNA to block Sema3fa production, and analyzed embryos throughout retinal development. Histological sections, in situ hybridization for retinal polarity markers and analysis of the retinal ganglion cells (RGCs) using the Tg(isl2b::GFP) reporter line were used to characterize the Sema3fa deficient embryos. We find that sema3fa is expressed around the choroid fissure and temporal retina prior to fissure closure and RGC axonal exit in wildtype embryos. Sema3fa deficient embryos present with coloboma of the eye at 48 hours post fertilization, defective patterns in the expression of retina polarity markers, and proliferation deficits in the temporal retina. During RGC axonogenesis, embryos present with aberrant axonal sprouts into the neural retina from the ganglion cell layer and optic nerve. Overall, these data suggest Sema3f as a novel factor involved in eye and choroid fissure morphogenesis, progenitor behaviour and neural retina development.