The behavior of the semiquinone anion radical of hypocrellin B (HB), produced by electrolysis, has been studied by cyclic voltammetry, steady-state electron spin resonance, time-resolved electron spin resonance and in situ UV-Vis spectra. The results showed that the formation mechanisms of HB-. were different between electrolysis and photolysis, and the disproportionation of semiquinone anion radical of HB (HB-.) could not occur in dimethylsulfoxide; on the contrary, a syn-proportionation reaction in which HB2- reacts with HB to form HB-. was observed experimentally. At the same time, the kinetic parameters of HB-. were obtained from the time-decay experiments of HB-..