Diabetic nephropathy (DN) endangers health and is a high financial public burden worldwide. Risk of DN is positively correlated with high levels of reactive oxygen species (ROS). Carnosine, an antioxidant, actively regulates cell function and has the potential to reduce the occurrence of DN. Here, we explored whether carnosine could prevent oxidative stress in human kidney tubular epithelial (HK2) cells and, if so, the mechanisms underlying this effect. HK2 cells were cultured with the ROS hydrogen peroxide (H2O2) for 24 h and then treated with carnosine. In H2O2-damaged HK2 cells, carnosine significantly increased cell viability, assessed using a Cell Counting Kit 8, increased total superoxide dismutase (TSOD) activity, assessed using a T-SOD activity detection kit, but decreased ROS levels, assessed using a ROS-sensitive fluorescent probe. Western blotting analyses to determine the protein expression levels of BAX, BCL-2, caspase 3, and the NADPH oxidase isoforms NOX2 and NOX4, as well as confocal laser scanning microscopy to assess changes in the mitochondrial membrane potential and the relative position of mitochondria to cytochrome c, indicated that carnosine inhibited apoptosis via the mitochondrial pathway in H2O2-damaged HK2 cells. Significantly decreased NOX4 expression and increased T-SOD activity in the presence of carnosine reduced the production of intracellular ROS, relieving oxidative stress to inhibit apoptosis via the mitochondrial pathway. These findings provide molecular mechanistic insights underlying the effects of carnosine, particularly as a potential therapeutic in DN. (C) 2020 The Authors. Published by Elsevier Inc.