Diverse molecular species of sulfatide with differences in fatty acid lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase (V-ATPase)-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope matrix-assisted laser desorption ionization-mass spectrometry analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. Additionally, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-V-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy fatty acids, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.