Yellowing of broccoli is a crucial limiting factor for its commercial value and consumer acceptance during postharvest. In this study, the impacts of exogenous melatonin (MEL) on chlorophyll content and fluorescence, as well as ultrastructure and membrane lipid metabolism of chloroplasts in broccoli were investigated during postharvest. The results showed that MEL treatment (200 μmol L−1) maintained the chlorophyll content, chloroplast autofluorescence and integral structure, and reduced the level ofserotonin in the chloroplasts in broccoli. Also, MEL treatment inhibited the membrane lipid peroxidation of chloroplasts, as indicated by low levels of superoxide anion (O2-), hydrogen peroxide (H2O2) and malondialdehyde (MDA), and high levels of endogenous MEL. In addition, the stability and fluidity of chloroplast membranes were also better maintained in the treated broccoli via increasing the contents of phosphatidylglyceroland (PG), monogalactosyldiglyceride (MGDG), digalactosyldiglyceride (DGDG) and unsaturated fatty acids as well as decreasing saturated fatty acid content and the activities of lipoxygenase (LOX) and lipase (LPS). Thus, the application of MEL facilitated the maintenance of chloroplast integrity, thus contributing to yellowing postponement and the extension of the storage life of broccoli.