Natural ceramides (Cers) possess a trans double bond between C4 and C5 of the sphingoid chain. This double bond is critical to their cell signaling properties. Both a change from trans to cis and the saturation of this site lead to changes in or loss of biological activity. To explore the conformational impact of the cis double bond, through-bond, and through-space interactions were investigated in hydrated Cers by multidimensional (1)H and (13)C NMR spectroscopy. Unlike trans-Cer, the cis-isomer exhibited not one but two broad yet resolved resonances for the protons in C1-OH and C3-OH, much like dihydroceramide (DHCer). Temperature-dependent studies and partial isotopic labeling of cis-Cer revealed that relative to trans-Cer, these two OH groups form weaker hydrogen bonds, particularly in the case of C1-OH. Our results also suggest that the cis double bond twists, slightly, the orientation of HO-C1 with respect to HO-C3, thus weakening the hydrogen-bonding network formed between the two OH groups of cis-Cer and bound water molecules. The alteration of the local network of H-bonds may account for the differences observed in the biological activity of the two isomers.