Hydrogen peroxide (H2O2), a major reactive oxygen species, has been shown to be a critical mediator of apoptosis induced by several toxic metals such as cadmium. In this study, we used the freshwater crab Sinopotamon henanense to study whether H2O2 can cause apoptosis in gill cells. The crabs were incubated in H2O2 and the DNA fragmentation, ultrastructural changes and caspase-3/8/9 activities were measured. The results showed that in freshwater crab, H2O2 was found to induce apoptosis, as confirmed by DNA fragmentation analysis and morphological observation of transmission electron microscopy. This apoptosis occurs in a concentration-dependent pattern. During the apoptotic process, caspase-3, caspase-8 and caspase-9 were activated by H2O2. In addition, multiple physiological and pathological changes of gill cells were discovered after 24 h exposure to 5 mM H2O2, including aggregation and condensation of nuclear chromatin, appearance of extremely irregular nuclei with finger-like buds, disappearance of the organelles around the nuclei, swollen and dissolved cristae of mitochondria. We propose that H2O2-induced stress leads to mitochondria lesions oxidative injury and triggers apoptotic response through the caspase pathway in freshwater crab.