The brain relies on interhemispheric communication for coherent integration of cognition and behavior. Surgical disconnection of the two cerebral hemispheres has granted numerous insights into the functional organization of the corpus callosum (CC) and its relationship to hemispheric specialization. Today, technologies exist that allow us to examine the healthy, intact brain to explore the ways in which callosal organization relates to normal cognitive functioning and cerebral lateralization. The CC is organized in a topographical manner along its antero-posterior axis. Evidence from neuroimaging studies is revealing with greater specificity the function and the cortical projection targets of the topographically organized callosal subregions. The size, myelination and density of fibers in callosal subregions are related to function of the brain regions they connect: smaller fibers are slow-conducting and connect higher-order association areas; larger fibers are fast-conducting and connect visual, motor and secondary somotosensory areas. A decrease in fiber size and transcallosal connectivity might be related to a reduced need for interhemispheric communication due, in part, to increased intrahemispheric connectivity and specialization. Additionally, it has been suggested that lateralization of function seen in the human brain lies along an evolutionary continuum. Hemispheric specialization reduces duplication of function between the hemispheres. The microstructure and connectivity patterns of the CC provide a window for understanding the evolution of hemispheric asymmetries and lateralization of function. Here, we review the ways in which converging methodologies are advancing our understanding of interhemispheric communication in the normal human brain.