p14ARF is a tumour suppressor which plays a critical role in p53-dependent or -independent cell growth control. Several studies have recently provided evidence that p14ARF can also interfere either directly or indirectly with some components of the RB signalling pathway to mediate its antiproliferative activity. The aim of this study was to explore the existence of direct relationships between p14ARF and RB proteins. We show that p14ARF promotes the accumulation of a hypoacetylated RB protein, when it is upregulated in a model of stable-inducible clones or physiologically induced following cell exposure to cytotoxic agents. Looking for the mechanisms involved in this process, we demonstrate that the histone acetyl transferase Tip60 directly interacts with RB and stimulates its degradation by the proteasome through acetylation of its C-terminus. Furthermore, and consistent with p14ARF-induced RB accumulation, we provide evidence that p14ARF prevents Tip60-mediated RB acetylation, therefore precluding its proteasomal degradation. Overall, our results identify a novel mechanism by which p14ARF controls the RB pathway to trigger its antiproliferative function.