The microscopic hydration of the ceramide headgroup has been determined using a combination of experimentalboth NMR and neutron diffraction techniques and computational techniquesempirical potential structure refinement (EPSR) and molecular dynamics (MD). The addition of water to ceramide in chloroform solutions disrupts the chloroform solvation of the ceramide headgroup, and the water forms distinct pockets of density. Specifically, water is observed to preferentially hydrate the two hydroxyl groups and the carbonyl oxygen over the amide NH motif. Further assessment of the location and orientation of the water molecules bound to the ceramide headgroup makes it clear that the strongly solvated carbonyl moiety of the amide bond creates an anchor from which water molecules can bridge via hydrogen bonding interactions to the hydroxyl groups. Moreover, a significant difference in the hydration of the two hydroxyl groups indicates that water molecules are associated with the headgroup in such a way that they bridge between the carbonyl motif and the nearest neighbor hydroxyl group.