Background: Despite the fundamental importance of cell membrane microviscosity, changes in this biophysical parameter of membranes during photodynamic therapy (PDT) have not been fully understood. Methods: In this work, changes in the microviscosity of membranes of live HeLa Kyoto tumor cells were studied during PDT with KillerRed, a genetically encoded photosensitizer, in different cellular localizations. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive BODIPY2 rotor. Results: Depending on the localization of the phototoxic protein, different effects on membrane microviscosity were observed. With nuclear localization of KillerRed, a gradual decrease in microviscosity was detected throughout the entire observation period, while for membrane localization of KillerRed, a dramatic increase in microviscosity was observed in the first minutes after PDT, and then a significant decrease at later stages of monitoring. The obtained data on cell monolayers are in good agreement with the data obtained for 3D tumor spheroids. Conclusions: These results indicate the involvement of membrane microviscosity in the response of tumor cells to PDT, which strongly depends on the localization of reactive oxygen species attack via targeting of a genetically encoded photosensitizer.