The production of ROS in mitochondria plays critical role in photodynamic therapy (PDT). The aim of this study was to investigate whether fullerene-based PDT can induce generation of additional endogenous ROS in mitochondria. Chitosan oligosaccharide grafted fullerene conjugate (CS-C-60) was synthesized as a model water-soluble fullerene. The relationship among photodynamic cytotoxicity, intracellular ROS and CS-C-60 amount demonstrated that low dose fullerene could induce generation of endogenous ROS in human malignant melanoma (A375) cells. Laser scanning microscope (LSM) image shows that considerable amount of endogenous ROS was generated in mitochondria even CS-C-60 could not localize into mitochondria. Assay with rotenone shows that PDT-induced endogenous ROS was generated via electron transport chain (ETC).