Determining atomistic structures of grain boundaries (GBs) is essential to understand structure–property interplay in oxides. Here, different GB superstructures in CuO nanosheets, including (111) and (114) twinning boundaries (TBs) and (002)/(223) GB, are investigated. Unlike the lower-energy stoichiometric (111) TB, both experimental and first-principles investigations reveal a severe segregation of Cu and O vacancies and a nonstoichiometric property at (114) TB, which may facilitate ionic transportation and provide space for elemental segregation. More importantly, the calculated electronic structures have shown the increased conductivity as well as the unanticipated magnetism in both (114) TB and (002)/(223) GB. These findings could contribute to the race towards the property-directing structural design by GB engineering.