The present study was designed to present evidence to clarify the relationships between learning ability, neuronal cell adhesion molecule L1 expression and hippocampal structural changes in the rat model received X-irradiation at an embryonic stage (E15). Water maze task indicated that all of the irradiated rats failed to learn the task in the whole training procedure. Their latency to the platform and swimming distance were significant differences from those sham-treated controls. Histological studies showed that the hippocampal ectopias induced by X-rays in the CA1 were involved in the spatial learning impairment, in which they hampered normal processes in learning development and transmission of information. Number, size and positions of the ectopias in the dorsal parts of the hippocampus were confirmed to be related to degrees of spatial learning impairment. On the other hand, L1 expression in the hippocampus was examined with Western blot analysis. The results indicated a lower content of L1 in the irradiated rats. A decrease in L1 might be one of reasons to cause disorganization of the septohippocampal pathways. These findings suggest some mechanisms of spatial learning impairment can be attributed to the formation of the hippocampal ectopias and redaction of L1 following prenatal exposure to X-irradiation.