ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces conformational changes in Msh2-Msh6; however, the nature of these conformational changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a conformational change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg(1124). An engineered disulfide bond within this region prevented the ATP-driven conformational change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven conformational change plays a role in mismatch repair.