Hepatocellular carcinoma suppressor 1 (HCCS1) was discovered as a novel tumor suppressor gene. We recently observed that adenovirus-mediated gene transfer of HCCS1 leads to cytotoxicity to human hepatocarcinoma cells. Here, we have demonstrated that adenovirus-mediated overexpression of HCCS1 induces apoptosis in hepatocarcinoma cells and have further characterized the apoptotic cascade. The results showed that lysosomal cathepsin D is released into the cytosol in response to HCCS1 overexpression and consequently triggers Bax insertion into the mitochondrial membrane, which leads to the release of cytochrome c. In addition, HCCS1 overexpression can induce an increase in intracellular free Ca(2+) concentration, which also results in cytochrome c release. The released cytochrome c activates downstream caspases, leading to the occurrence of the late stages of apoptosis. Moreover, we demonstrated that the disruption of HCCS1 in mice leads to embryonic lethality, accompanied by abnormal labyrinth architecture resulting from the excessive proliferation of trophoblast cells in the placenta. These results suggest that HCCS1 plays a role in apoptosis regulation and development.