We investigate the killing effect of photodynamic therapy (PDT) induced by photocarcinorin (PsD007) on human epidermoid carcinoma Hep-2 in vitro, and explore the preliminary killing mechanism. Methyl thiazolyl tetrazolium (MTT) colorimetric assay is applied to measure the relative survival rate of PDT with different photosensitizer concentrations, light doses and antioxidants. Confocal microscope is used to observe the subcellular localization of PsD007. Annexin V/PI double staining is used to detect the apoptosis rate. DCFH-DA probe is used the to detect reactive oxygen species (ROS) production. The fatality rate of PDT on Hep-2 can be as high as 85.4%, and is positively correlated with photosensitizer concentration and energy density. Three kinds of antioxidants can all antagonize the damage effect induced by PDT in different degree, while sodium azide has the best effect. The fluorescence images of PsD007 subcellular localization demonstrate that PsD007 diffuses into the mitochondria. Two hours after PDT, the ROS production is as 2.71 times as control group. The apoptosis rate is up to 49.5% (energy density is 1.2 J/cm2) and 70.2% (energy density is 4.8 J/cm2) four hours after PDT. PsD007-PDT could significantly kill Hep-2 cells and induce apoptosis that mitochondria may be the initial target.