The major source of endogenous hydrogen peroxide is generally thought to be the respiratory chain of bacteria and mitochondria. In our previous works, mesosome structure was induced in cells during rifampicin effect, and the mesosome formation is always accompanied by excess hydrogen peroxide accumulation in bacterial cells. However, the underlying mechanisms of hydrogen peroxide production and the rationale behind it remain still unknown. Here we report that hydrogen peroxide can specifically accumulate in the mesosome in vitro. Mesosomes were interpreted earlier as artifacts of specific cells under stress through TEM preparation, while, in the current study, mesosomes were shown as intracellular compartments with specific roles and features by using quickly freezing preparation of TEM. Formation of hydrogen peroxide was observed in suspension of mesosomal vesicles by using either a fluorescence-based reporter assay or a histochemical method, respectively. Our investigation provides experimental evidence that mesosomes can be a novel site of hydrogen peroxide accumulation.