Many residues within proteins adopt conformations that appear to be stabilized by interactions between an amide N-H and the amide N of the previous residue. To explore whether these interactions constitute hydrogen bonds, we characterized the IR stretching frequencies of deuterated variants of proline and the corresponding carbamate, as well as the four proline residues of an Src homology 3 domain protein. The CδD2 stretching frequencies are shifted to lower energies due to hyperconjugation with Ni electron density, and engaging this density via protonation or the formation of the Ni+1-H···Ni interaction ablates this hyperconjugation and thus induces an otherwise difficult to explain blue shift in the C-D absorptions. Along with density functional theory calculations, the data reveal that the Ni+1-H···Ni interactions constitute H-bonds and suggest that they may play an important and previously underappreciated role in protein folding, structure, and function.