Hyperhydric disorders occur frequently in plant tissues cultured in vitro and cause several morphological and physiological abnormalities. However, a systematic defense response is triggered by hyperhydric conditions. The accumulation of reactive oxygen species (ROS), activities of antioxidant enzymes and their immunoblots, and the proteome-level changes in normal versus hyperhydric shoots of carnation (Dianthus caryophyllus) cultured in vitro were investigated. Total proteins were also extracted from the shoot and analyzed by two-dimensional electrophoresis. Among a total of 700 spots detected, only 40 had significant changes in abundance in the hyperhydric compared to the normal shoots, which were further identified by a mass spectrometer (MALDI-TOF MS). Most of them were involved in photosynthesis, RNA processing, and general metabolisms, while the rest were involved in secondary metabolic processes. These identified proteins in carnation shoots may provide novel evidences for stress tolerance against hyperhydricity. © 2016, Springer Science+Business Media New York.