Di-cysteine substituted hypocrellin B (DCHB) is a new water-soluble photosensitizer with significantly enhanced red absorption at wavelengths longer than 600 nm over the parent compound hypocrellin B (HB). The photosensitizing properties (Type I and/or Type II mechanisms) of DCHB have been investigated in dimethylsulfoxide (DMSO) and aqueous solution (pH 7.4) using electron paramagnetic resonance (EPR) and spectrophotometric methods. In anaerobic DMSO solution, the semiquinone anion radical of DCHB (DCHB-) is predominantly photoproduced via self-electron transfer between excited- and ground-state DCHB species. The presence of an electron donor significantly promotes the formation of the reduced form of DCHB. When a deoxygenated aqueous solution of DCHB and an electron door are irradiated with 532 nm light, the hydroquinone of DCHB (DCHBH2) is formed via the disproportionation of the first-formed DCHB- and second electron transfer to DCHB- and second electron transfer to DCHB- from the electron donor. When oxygen is present, singlet oxygen (1 O2), superoxide anion radical (O2-) and hydroxyl radical (OH) are produced. The quantum yield of 1 O2 generation by DCHB photosensitization is estimated to be 0.54 using Rose Bengal as a reference, a little lower than that of HB (0.76). The superoxide anion radical is also significantly enhanced by the presence of electron donors. Moreover, O2- upon disproportionation generated H2O2 and ultimately the highly reactive OH via the Haber-Weiss reaction pathway. The efficiency of O2- generation by DCHB is obviously enhanced over that of HB. These findings suggest that the photodynamic actions of DCHB may proceed via type I and Type II mechanisms and that this new photosensitizer retains photosensitizing activity after photodynamic therapy-oriented chemical modification.