We have investigated the interaction between hydrogen isotopes and defects in Li2O, using Fourier transform infrared absorption spectroscopy (FT-IR). Multiple peaks were observed in the O–D stretching vibration region with Li2O single crystals which were treated by thermal absorption and quenching. These peaks had different dependence on temperature and were attributable to the stretching vibrations of O–D in bulk Li2O with or without defects. We have also studied the nature of hydrogen isotopes in Li2O with defects by the ab-initio quantum chemical calculation technique. The influence of defects on hydroxyl groups is discussed.