Developing the cell-impermeable Ru(II) polypyridyl cationic complexes as effective photosensitizers (PS) which have high cellular uptake and photo-toxicity, but low dark toxicity, is quite challenging. Here we found that the highly reactive singlet oxygen (¹O₂) can be generated by the irradiation of a typical Ru(II) polypyridyl complex Ru(II)tris(tetramethylphenanthroline) ([Ru(TMP)₃]²⁺) under visible light irradiation by ESR with TEMPO (2,2,6,6-tetramethyl-4-piperidone-N-oxyl) as ¹O₂ probe. Effective cellular and nuclear delivery of cationic [Ru(TMP)₃]²⁺ was achieved through our recently developed ion-pairing method, and 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP) was found to be the most effective among all chlorophenols tested. The accelerated cellular, especially nuclear uptake of [Ru(TMP)₃]²⁺ results in the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and DNA strand breaks, caspase 3/7 activation and cell apoptosis in HeLa cells upon light irradiation. More importantly, compared with other traditional photosensitizers, [Ru(TMP)₃]²⁺ showed significant photo-toxicity but low dark toxicity. Similar effects were observed when 2,3,4,5-TeCP was substituted by the currently clinically used anti-inflammatory drug flufenamic acid. This represents the first report that the cell-impermeable Ru(II) polypyridyl complex ion-paired with suitable lipophilic counter-anions functions as potent intracellular photosensitizer under visible light irradiation mainly via a ¹O₂-mediated mechanism. These findings should provide new perspectives for future investigations on other metal complexes with similar characteristics as promising photosensitizers for potential photodynamic therapy.