Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.