Reactive oxygen species (ROS) are an important factor in the development of skin lesions in diabetes. A new antioxidant, hydrogen, can selectively neutralize hydroxyl radicals ((OH)-O-center dot) and peroxynitrite (ONOO-) in cell-free systems, whereas it seldom reacts with other ROS. Fibroblasts are a key component of skin. In the present study, we investigated the protective effects of hydrogen-rich medium on human skin fibroblasts (HSFs) under oxidative stress. Confocal microscopy was used to assay both the intracellular superoxide anion (O-2(-)) concentration and the mitochondrial membrane potential (Delta Psi). Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular malonaldehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) were also measured. The results revealed that both mannitol and high glucose could cause oxidative stress in HSFs. Interestingly, the use of a hydrogen-rich medium significantly reduced the level of intracellular O-2(-). stabilized the Delta Psi and attenuated production of MDA, 8-OHdG and 3-NT which efficiently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage. In other words, hydrogen decreased the excessive generation of intracellular O-2(-) and elevated the cellular antioxidative defense. Based on our results, hydrogen may have applications in the treatment of skin diseases caused by diabetes. (C) 2011 Elsevier Inc. All rights reserved.