The subcommissural organ (SCO) is a roof plate differentiation located in the caudal diencephalon under the posterior commissure (PC). A role for SCO and its secretory product, SCO-spondin, in the formation of the PC has been proposed. Here, we provide immunohistochemical evidence to suggest that SCO is anatomically divided in a bilateral region positive for SCO-spondin that surrounds a negative medial region. Remarkably, axons contacting the lateral region are highly fasciculated, in sharp contrast with the defasciculated axons of the medial region. In addition, lateral axon fascicles run toward the midline inside of tunnels limited by the basal prolongations of SCO cells and extracellular SCO-spondin. Our in vitro data in collagen gel matrices show that SCO-spondin induces axonal growth and fasciculation of pretectal explants. Together, our findings support the idea that SCO-spondin participates in the guidance and fasciculation of axons of the PC.