CLIC4/mtCLIC (referred to here as CLIC4) is one of the seven-member family of chloride intracellular channels (CLIC). CLIC4 localizes to the mitochondria, nucleus, cytoplasm and other organellular compartments and participates in the apoptotic response to stress. However, the role of CLIC4 in oxidative stress and apoptosis is not well understood. In this study, we showed the important role of CLIC4 in apoptosis of C6 glioma cells induced by hydrogen peroxide (H2O2). Our results showed that CLIC4 protein expression was upregulated following H2O2-induced C6 cell apoptosis. The upregulation of CLIC4 protein expression was paralleled with an increased Bax/Bcl-2 ratio, cytochrome c and cleaved caspase-3 protein expression upon H2O2-induced C6 cell apoptosis. Suppression of CLIC4 expression by RNA interference enhanced cell apoptosis, but the ratio of Bax/Bcl-2 was not involved in this process. Dissipation of mitochondrial membrane potential and nuclear translocation of CLIC4 were involved in the activation of apoptosis induced by H2O2. Our data indicate that CLIC4 protein may be a key element in the apoptotic response to oxidative stress.