The water-soluble Roussin's red ester [(NO)₂Fe(µ-SCH₂CH₂P(O)(CH₂OH)₂)₂Fe(NO)₂] (1), a potential photochemical prodrug of an NO precursor, was synthesized from the reaction of HSCH₂CH₂P(O)(CH₂OH)₂ (F) and [Fe(CO)₂(NO)₂]. The IR v(NO) stretching frequencies of complex 1 appear at 1759 (s), 1784 (s) and 1816 (w) cm⁻¹ in buffer (pH = 7.4). NO was released with a stoichiometry ratio Δ[NO]/Δ[1] = 3.6 ± 0.2 when complex 1 was exposed to UV in deaerated aqueous phosphate buffer solution. Here light acts as an On/Off switch for NO release. Incubation of pBR322 supercoiled DNA with complex 1, followed by irradiation, produced DNA strand breakage. In contrast to the addition of carboxy-PTIO (NO radical scavenger), DNA strand breakage was not inhibited when the scavengers of hydroxyl radical and singlet oxygen were added. Complex 1 irradiated under a N₂ atmosphere exhibited the same cleavage efficiency as complex 1 irradiated under air. The results show that DNA strand cleavage efficiency depends on the concentration of complex 1, the pH value of the buffer, and the duration of the photolysis of complex 1. The conversion rate from supercoiled (SC form) to nicked circular (NC form) of complex 1 was 2.96 × 10⁻² s⁻¹. The results of a T4 ligase enzymatic assay reveals the nonhydrolytic DNA breakage mechanism. The NO-release ability of complexes 1, 2, and 3 follows the order 1 > 2 > 3. Upon UV-irradiation, complex 1 exhibits cytotoxicity against B16-F10 mouse melanoma cells.