Granzyme B (GrB) is an important serine protease involved in granule-mediated killing in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In order to study whether ectopic expression of GrB in tumor cells can induce cell death, expression vectors encoding active GrB (GrBa) and mutant GrBa (mGrBa) gene in which serine in catalytic triad was replaced with cysteine were constructed, and transiently transfected into HeLa cells with lipofectamine. It was shown by GFP coexpression, indirect immunofluorescence, cell counting and MTT analyses that ectopic expression of GrBa genes caused increased tumor cell size and multinucleation, and the growth of cells that expressed GrBa proteins was inhibited. Retarded growth was further observed in these morphologically abnormal cells isolated by 40% percoll, which directly contributed to growth inhibitory effect on GrBa-transfectants. There was cytoskeletal breakdown and abnormal mitosis characteristic of multiple spindle poles, largely resulting in accumulation of giant multinuclear cells. These results suggest that GrBa may serve as a good candidate in tumor gene therapy.