Though cytoplasmic male sterility (CMS) in peppers is associated with the orf507 gene, definitive and direct evidence that it directly causes male sterility is still lacking. In this study, differences in histochemical localization of anther cytochrome c oxidase between the pepper CMS line and maintainer line were observed mainly in the tapetal cells and tapetal membrane. Inducible and specific expression of the orf507 gene in the pepper maintainer line found that transformants were morphologically similar to untransformed and transformed control plants, but had shrunken anthers that showed little dehiscence and fewer pollen grains with lower germination rate and higher naturally damaged rate. These characters were different from those of CMS line which does not produce any pollen grains. Meanwhile a pollination test using transformants as the male parent set few fruit and there were few seeds in the limited number of fruits. At the tetrad stage, ablation of the tapetal cell induced by premature programmed cell death occurred in the transformants and the microspores were distorted and degraded at the mononuclear stage. Stable transmission of induced semi-male sterility was confirmed by a test cross. In addition, expression of orf507 in the maintainer lines seemed to inhibit expression of atp6-2 to a certain extent, and lead to the increase of the activity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATP synthase. These results introduce the premature programmed cell death caused by orf507 gene in tapetal cells and semi-male sterility, but not complete male sterility.