Abstract: Objective：To observe photodamage effect of riboflavin on Hela cells, and preliminarily explore its subcellular localization. Methods：The survival rate of Hela cells without or with illumination (365 nm, 45 min) treated by 10, 50, 100, 150, and 250 μmol/L riboflavin was observed using the MTT method. The subcellular localization of riboflavin with above concentrations in Hela cells was observed under fluorescence microscope. The morphological change of Hela cells was observed in single riboflavin (100 μmol/L), single illumination (365 nm, 45 min), riboflavin (100 μmol/L) plus illumination (365 nm, 45 min) , and normal conditions under optical microscope. Results：Compared with the normal group, the survival rate of Hela cells was not changed by riboflavin co-incubation with above concentrations in non-illumination condition. However, the survival rate of Hela cells was reduced by riboflavin co-incubation under illumination (P<0.05). Fluorescence microscopy results showed that the riboflavin localized mainly in the perinuclear area and nuclear membrane when its concentration was low (25 μmol/L), and also localized in the cytoplasmic membrane when its concentration was high (200 μmol/L). Conclusions：The riboflavin could induce photodamage to Hela cells, the subcellular localization involves both cell nucleus and cell membrane, which is a comprehensive damage mechanism.