The corpus callosum (CC) is the largest interhemispheric commissure of the mammalian brain, and it includes axons, cortical neurons, and glial cells. It is mainly composed of myelin, a lipidic sheath which is produced by glial cell membranes; myelin is wrapped up around axons and plays a fundamental role in the fast conduction of neuronal electrical signals. The human CC is divided into various anatomical regions, with different axonal composition, including, from front to back, genu, body or trunk, isthmus, and splenium. Corpus callosum undergoes some alterations not only in the presence of specific physiological and pathological conditions, but also because of aging. For the first time, in the present study a hyperspectral imaging analysis of human corpus callosum was performed. The study, carried out on CC autopsy samples collected from human adult males of different ages, was focused mainly on the genu and splenium regions. By combining Fourier-transform infrared imaging and histological analyses with multivariate and univariate ones, the macromolecular composition of these regions was defined, and age-related alterations in the lipid and protein components were identified.