Ras association (RalGDS/AF-6) domain family member RASSF5 is a non-enzymatic RAS effector super family protein, known to be involved in cell growth regulation. Expression of RASSF5 is found to be extinguished by promoter hypermethylation in different human cancers, and its ectopic expression suppresses cell proliferation and tumorigenicity. Interestingly, this role in tumorigenesis has been confounded by the fact that regulation at molecular level remains unclear and many transformed cells actually display elevated RASSF5 expression. Here, we demonstrate that E3 ubiquitin ligase Itch is a unique binding partner of RASSF5. Itch can interact with PPxY motif in RASSF5 both in vivo and in vitro through its WW domains. Importantly, the overexpression of Itch induces RASSF5 degradation by poly-ubiquitination via 26S proteasome pathway. In addition, our results indicate that the elevated levels of RASSF5 found in tumor cells due to acetylation, which restricts its binding to Itch and results in a more stable inert protein. Inhibition of RASSF5 acetylation permits its interaction with Itch and provokes proteasomal degradation. These data suggest that apart from promoter methylation, hyperacetylation could also be downregulating RASSF5 function in different human cancer. Finally, results from functional assays suggest that the overexpression of wild type, not the ligase activity defective Itch negatively regulate RASSF5-mediated G1 phase transition of cell cycle as well as apoptosis, suggesting that Itch alone is sufficient to alter RASSF5 function. Collectively, the present investigation identifies a HECT class E3 ubiquitin ligase Itch as a unique negative regulator of RASSF5, and suggests the possibility that acetylation as a potential therapeutic target for human cancer.