Class 3 semaphorins are known to repel and/or sometimes attract axons; however, their role in guiding developing axons in the CNS in vivo is still essentially unknown. We investigated the role of Semaphorin3D (Sema3D) in the formation of the early axon pathways in the zebrafish CNS. Morpholino knock-down shows that Sema3D is essential for the correct formation of two early axon pathways. Sema3D appears to guide axons of the nucleus of the medial longitudinal fasciculus (nucMLF) by repulsion and modulation of fasciculation. In contrast, Sema3D appears to be attractive to telencephalic neurons that form the anterior commissure (AC). Knock-down of Neuropilin-1A (Npn-1A) phenocopied the effects of Sema3D knock-down on the nucMLF axons, and knock-down of either Npn-1A or Npn-2B phenocopied the defects of the AC. Furthermore, simultaneous partial knock-down experiments demonstrated genetic interactions among Sema3D, Npn-1A, and Npn-2B. Together, these data support the hypothesis that Sema3D may act as a repellent through receptors containing Npn-1A and as an attractant via receptors containing Npn-1A and Npn-2B.