The photodynamic properties of meta-tetra(hydroxy-phenyl)chlorin (mTHPC), a promising second-generation photosensitizer, were investigated using a human colon adenocarcinoma cell line (Colo 201 cells). The study on photocytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that mTHPC was an effective photosensitizer on Colo 201 cells. The photocytotoxicity of mTHPC showed both drug and light dose–dependent characteristics. To reach LD₅₀, namely, the dose at which 50% of the cells were killed, only 0.45 ± 0.15 μg/mL of mTHPC and 3 J/cm² of light dose were required. The presence of 10% fetal calf serum in culture medium significantly decreased the incorporation of mTHPC into cells and resulted in the reduction of photodynamic efficacy. Using confocal laser scanning microscopy, mTHPC was first shown to localize in lysosomes rather than in mitochondria. Furthermore, nuclear stainings demonstrated that photodynamic therapy with mTHPC induced apoptosis in Colo 201 cells.