The mechanism of DNA damage induced by photoexcited rhodamine analogues was studied using ³²P-5’-end-labeled DNA fragments. As photosensitizers, rhodamine-6G, rhodamine-110, and rhodamine-123 were used. Rhodamine-6G photosensitized DNA damage under aerobic or anaerobic condition, whereas rhodamine-110 and rhodamine-123 induced no or very weak DNA photodamage. Photo-irradiated rhodamine-6G caused DNA cleavage specifically at every guanine residues, when the DNA fragments were treated with piperidine or E. coli formamidopyridine DNA glycosylase. These treatments can induce the excision of the 2,6-diamino-4-hydroxy-5-formamidopyrimidine residues of DNA. Trace amount of 8-oxo-7,8-dihydro-2’-deoxyguanosine, an oxidized product of 2’-deoxyguanosine, was generated by photoexcited rhodamine-6G. A possible mechanism of DNA photodamage by rhodamine-6G is the guanine modification via the electron transfer rather than the generation of reactive oxygen species. In conclusion, rhodamine-6G can photosensitize guanine-specific DNA damage through oxygen-independent mechanism.