The influence of mannose treatment on mitochondrial physiological parameters was investigated to postpone broccoli senescence. The results showed that mannose treatment could reduce the level of mitochondrial reactive oxygen species (ROS) to slow down the damage to its ultrastructure. Mannose inhibited malondialdehyde (MDA) accumulation and the decline of cytochrome c/a (Cyt c/a), and delayed the increase of mitochondrial membrane permeability. It also alleviated the over-opening of the mitochondrial membrane permeability transition pore (MPTP) by regulating expressions of MPTP component genes such as BoVDAC, BoHXK and BoANT. Moreover, mannose treatment improved the activities of mitochondrial antioxidant enzymes, including ascorbate oxidase (APX), superoxide dismutase (SOD), catalase (CAT), and ascorbic acid (AsA) content. Also, it increased the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and Ca2+-ATPase, which were crucial for the mitochondrial energy metabolism. Taken together, our data indicated that mannose treatment inhibited the senescence of broccoli by maintaining the mitochondrial morphological structure and function.