Objective To investigate the photodynamic antitumor activity and chemical characteristics of pheophorbide A (PPBa)in vitro. Methods Breast cancer cells (MCF-7), lung cancer cells (A549), cervical cancer cells (HeLa), and three kinds of hepatoma cells (HepG2, hep3B and sk-Hep1) were planted in 96-well plates.The effects of light and dark toxicity, light intensity, and drug concentration on the phototoxicity of PPBa were investigated by MTT, and the uptake of PPBa was observed by Hoechst staining under a confocal microscope.The production of singlet oxygen was observed by flow cytometry and confocal microscopy with the reactive oxygen species detection kit.The photobleaching of PPBa was investigated by measuring the absorbance by a microplate reader according to the luminescence characteristics of PPBa. Results PPBa showed strong phototoxicity and low dark toxicity to six kinds of cancer cells, and IC₅₀ values to cancer cells were as follows: MCF-7:1.033 μmol/L, A549:1.911 μmol/L, HeLa: 2.319 μmol/L, HepG2:2.015 μmol/L, Hep3B: 2.089 μmol/L, sk-Hep1:2.467 μmol/L.The main uptake site of PPBa was the cytoplasm.The production of singlet oxygen was strongly dependent on the administration concentration, and photobleaching was very low. Conclusion For the six kinds of cancer cells, PPBa has the highest phototoxicity to breast cancer cells (MCF-7), with excellent properties and ideal photosensitizer characteristics.