Photo-oxidation is a potential pathway for the transformation of Cr(III) to Cr(VI) in natural environments. In this study, the Cr(III)-citrate complex (Cr(III)-cit) was prepared and its speciation was determined by high performance liquid chromatography (HPLC). Results showed that Cr(III)-cit existed in [Cr(III)-H-cit](+) and [Cr(III)-cit] species in a pH range of 3-5, in [Cr(III)-cit] only from pH 6-8, in [Cr(III)-cit] and [Cr(III)-OH-cit](-) from pH 9-11, and only in [Cr(III)-OH-cit](-) at pH 12. Additional experiments were conducted in batch systems with pHs of 5 to 12 at 25 °C, where aqueous Cr(III) and Cr(III)-cit were fully exposed to light from medium pressure mercury lamps and a xenon lamp mimicking solar light irradiation. Results demonstrated that oxidation of Cr(III) in Cr(III)-cit was much faster than that in aqueous Cr(III). Rates of Cr(III) photo-oxidation were not sensitive to pH in the range from 7 to 9 but increased significantly with further increases in pH, which was consistent with the distribution of Cr(III) forms. It appeared that [Cr(III)-cit-OH](-) was the most photochemically active form and Cr(II), resulting from a ligand-to-metal charge-transfer (LMCT) pathway after light absorption, was a precursor of the oxidation of Cr(III) to Cr(VI). Both dissolved oxygen and the hydroxyl radical ((•)OH), an intermediate, served as oxidants and facilitated the oxidation of Cr(II) to Cr(VI) via a multiple step pathway. The photoproduction of (•)OH was detected by HPLC using benzene as a probe, supporting the proposed reaction mechanism.