The hair is composed of the cuticle (the outermost surface), cortex (its major part), and medulla (in the hair center). The lipid content of the medulla of Caucasian hair is relatively higher than that of African-American hair. However, the types of lipids therein remain unidentified. The aim of the current study was to analyze the constituent lipids of the medulla of Japanese hair, and to identify their function. A lipid peroxidase fluorescent reagent was used to investigate region-specific differences in the lipid content of the medulla (the tip, middle, and root portions). Since the medulla is important for the hair's glossiness, we also investigated the relationship between the lipid content and hair glossiness. The lipid content of hair, based on the absence or presence of lipid species in the medulla, was investigated using thin-layer chromatography (TLC). Micro-attenuated total reflectance Fourier-transform infrared spectrophotometry (micro-ATR FTIR) was used to analyze the similarities between the standard and medulla lipids, focusing on the methylene/methyl stretching vibration region. The data indicated that the medulla contained unsaturated lipids, the content of which decreased from the root to the tip of the hair. Hair glossiness was reduced with the decreasing lipid content, suggesting that unsaturated lipids of the medulla play a role in glossiness. The TLC analysis revealed differences in the type and amount of hair lipids in the medulla. While squalene and oleic acid spots were detected in hair with a continuously maintained medulla, these compounds were not detected in hair in which the medulla was not maintained. The medulla constituents similar to oleic acid and squalene were also identified by the micro-ATR FTIR spectrum analysis and the previous report. The findings indicate that the medulla is composed of at least squalene and oleic acid.