BACKGROUND: Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by irradiating photosensitizers with light exposure to produce reactive oxygen species (ROS). An ideal photosensitizer is a crucial element to PDT. In the current study, we evaluated the photodynamic activity of a novel photosensitizer, the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB), both in vitro and in vivo. METHODS: Physicochemical characteristics of the novel photosensitizer were compared with that of its parent HB. The intracellular distribution of photosensitizers and mitochondrial membrane potential were detected with laser scanning confocal microscopy. The pathway of cell death was analyzed by flow cytometry. The release of proapoptotic proteins was evaluated by Western blot. S180 tumor model was used to evaluate the antitumor effects of PENHB-mediated PDT. RESULTS: Compared with its parent HB, water solubility of the derivative was improved enormously (6.6 mg/ml vs. 4.6 μg/ml), rendering its intravenous injection feasible without auxiliary solvent. The derivative had better PDT effect than HB in vitro under similar dark cytotoxicity. Moreover, PENSHB-mediated PDT was able to induce mitochondrial inner membrane permeabilisation, cytochrome c release, caspase-3 activation and subsequent apoptotic death. In vivo study showed that more than half of tumor bearing mice were cured by PENSHB-mediated PDT. CONCLUSIONS: In vitro and in vivo studies suggest that PENSHB is an effective photosensitizer for PDT to tumors. Therefore, PENSHB as a novel photosensitizer has a good prospect of clinical application.