Ammonia can cause cells to produce a large amount of reactive oxygen species (ROS), leading to the oxidative stress of cells. As the main intracellular reductant, nicotinamide adenine dinucleotide phosphate (NADPH) plays a crucial role in maintaining reduced glutathione (GSH), helping to remove ROS and protect cells from oxidative damage. Our study demonstrated that SIRT5 desuccinylated isocitrate dehydrogenase 2 (IDH2) to enhance its activity, resulting in increased NADPH production. Furthermore, we observed that SIRT5 overexpression alleviated ammonia-induced high levels of ROS in bovine mammary epithelial cells. This effect was achieved by activating IDH2 through SIRT5, which increased NADPH production and GSH levels, thereby improving the antioxidant capacity to scavenge ROS and reduce the susceptibility of cell to ROS. In conclusion, our findings revealed a SIRT5-dependent mechanism that modulated intracellular NADPH homeostasis to attenuate ammonia-induced oxidative stress by enhancing IDH2 enzymatic activity.