Caspy2, a zebrafish protease, is an active caspase for inducing apoptosis in mammalian cells. To investigate whether caspy2-mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid was mixed with cationic liposome and introduced into various tumour cell lines in vitro. Our data showed that caspy2 induced remarkable apoptosis of cancer cells in vitro. Treatment of mice-bearing CT26 colon carcinoma or MethA fibrosarcoma with intratumoral injection of liposome-caspy2 plasmid complex resulted in substantial killing of neoplastic cells and long-term survivors. Apoptotic cells were widely distributed in caspy2-treated tumour tissue. Infiltration of CD8(+) T lymphocyte was also observed apparently in the tumour tissue after the treatment with caspy2. Tumour-specific major histocompatibility complex (MHC) class I-dependent CD8(+) cytotoxic T lymphocyte activity was found by means of (51)Cr release assay. In MethA model, the mice acquired a long-time protective immunity against the parental tumour cell re-challenge. These results indicated that caspy2 can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumour effect. Furthermore, in vivo depletion of CD8(+) T lymphocytes could completely abrogate the antitumour immune activity, whereas the depletion of CD4(+) cells showed partial abrogation. In this study, we developed a novel anticancer strategy that combines both induction of apoptosis and immune response in mice-bearing tumours with a single caspy2 gene. This approach may provide an important way for treatment of cancer.