TIP60 is a tumor suppressor with histone acetyltransferase (HAT) activity, playing a crucial role in regulating chromatin architecture by acetylating histones to enhance accessibility for other regulatory factors. Its function is vital for several key cellular processes, including DNA damage repair, apoptosis, and autophagy. While the downregulation of TIP60 has been associated with various cancers, the effects of naturally occurring mutations in TIP60 on its function in malignancies remain poorly understood. In this study, we explored how cancer-related mutations in TIP60 impact its structure and function. Several deleterious and destabilizing missense mutations were identified and analyzed for structural changes. Molecular dynamics simulations revealed alterations in protein conformational stability and radius of gyration due to these mutations, supported by significant changes in TIP60's solvent accessibility and intramolecular hydrogen bonding. Biochemical assays with recombinant proteins showed a loss of catalytic activity in the G400W mutant. Live cell imaging indicated abnormal localization of the G400W mutant within the nucleus. Additionally, we observed aberrant phase separation of TIP60 caused by the G400W mutation. Notably, the G400W mutation impairs TIP60's catalytic function, preventing effective DNA repair and leaving the genome vulnerable to further mutations. Our findings highlight cancer-associated mutations in TIP60 that may contribute to the molecular mechanisms underlying cancer initiation and progression.