The influence of 90° domain boundaries in (La,Li)TiO3 (LLTO) on the Li conduction mechanism has been examined by a combination of state-of-the-art electron microscopy techniques and first-principles calculations. The atomistic structure of 90° domain boundaries in LLTO was determined from aberration-corrected scanning transmission electron microscopy images. At 90° domain boundaries, each perovskite unit of one domain is connected by an La-rich layer to units of the neighboring domain. First-principles calculations of a model domain boundary show that Li migration through the La layer has a very high activation energy, E a, of 3.58 eV, indicating that La layers serve to block Li migration. However, if La vacancies are present within La layers, the migration energy decreases significantly to 0.58 eV, a value more in line with experimental observation. The results show that Li conduction in LLTO is strongly influenced by 90° domain boundaries. The activation energy in a single crystal (E a = 0.19 eV) is much lower, suggesting that if 90° domain boundaries could be eliminated, an increase in conductivity of approximately three orders of magnitude compared with conventional LLTO could be achieved.