The impact of developmental ablation of Pax6 function on morphology and functional connectivity of the adult cerebrum was studied in cortex-specific Pax6 knockout mice (Pax6cKO) using structural magnetic resonance imaging (MRI), manganese-enhanced MRI, and diffusion tensor MRI in conjunction with fiber tractography. Mutants presented with decreased volumes of total brain and olfactory bulb, reduced cortical thickness, and altered layering of the piriform cortex. Tracking of major neuronal fiber bundles revealed a disorganization of callosal fibers with an almost complete lack of interhemispheric connectivity. In Pax6cKO mice intrahemispheric callosal fibers as well as intracortical fibers were predominantly directed along a rostrocaudal orientation instead of a left-right and dorsoventral orientation found in controls. Fiber disorganization also involved the septohippocampal connection targeting mostly the lateral septal nucleus. The hippocampus was rostrally extended and its volume was increased relative to that of the forebrain and midbrain. Manganese-induced MRI signal enhancement in the CA3 region suggested a normal function of hippocampal pyramidal cells. Noteworthy, several morphologic disturbances in gray and white matter of Pax6cKO mice were similar to observations in human aniridia patients. The present findings indicate an important role of Pax6 in the development of both the cortex and cerebral fiber connectivity.