Environmental transport of Tl is affected by redox reaction between Tl(I) and Tl(III) and ligand exchange reactions of them. In order to deepen the knowledge of Tl chemistry, we investigated fractionation of Tl stable isotopes (Tl-203 and Tl-205) in a chemical exchange system. Tl isotopes were fractionated in a liquid-liquid extraction system, in which aqueous and organic phases are hydrochloric acid solution and dichloroethane including a crown ether, respectively. After purification by ion-exchange chemistry, the isotope ratio of Tl-205/Tl-203 in equilibrated aqueous phase was measured precisely by multiple-collector-inductively-coupled-plasma-mass-spectrometry. A large isotope fractionation > 1 aEuro degrees was found. Electronic structures of possible Tl species (hydrated Tl+, Tl3+, and Tl chlorides) were calculated by ab initio methods, and the isotope fractionation factor was theoretically obtained. The isotope fractionation via intramolecular vibrations was calculated to be much smaller than the experimental result. The isotope fractionation via isotopic change in nuclear volume, named the nuclear field shift effect, was calculated to be > 1 aEuro degrees in Tl(I)-Tl(III) redox systems and/or ligand exchange systems of Tl(III). The nuclear field shift effect was found to be the major origin of Tl isotope fractionation.