The cytotoxic efficacy of methylene blue-sensitized photoinactivation of bladder cancer cells has been proved in vitro and in vivo in our previous work. The ultrastructural changes of MBT-2 murine bladder cancer cells following methylene blue-sensitized phototherapy have been evaluated by scanning and transmission electron microscopy. MBT-2 cells were pretreated with 0.1% methylene blue, and then exposed to ordinary light for different durations from 1 to 60 min. Both electron microscopic examinations showed that changes in tumor cells occurred at 5 min of illumination with fenestration and minipore formation on the cell membrane and mitochondrial disintegration. At 30 min, cleavage and cracking of the cell membrane with ribosomal lysis were noted. Finally, fragmentation with decomposition of the membrane and chromosomal condensation and lysis resulted at 60 min. This study showed that the cytotoxic effect induced by methylene blue phototherapy is initiated in the cell membrane and intracellular mitochondria in the early phase.