Histones and polyamines are important determinants of chromatin structure. Histones are the core of nucleosome particles and their modification by acetylation of N-terminal tails is involved in chromatin structural changes and transcriptional modulation. Polyamines also, including spermidine, are targets of both nuclear and cytoplasmic acetylation, which in turn alters their affinity for DNA and nucleosomes. Previous studies report an interplay existing between polyamines metabolism and levels, and histone acetylation. The relationship between polyamines and histone epigenetics in vivo has been considered in various models of ageing through epigenetic modifications, induction of autophagy and suppression of necrosis, but the molecular basis of this effect is still unclear. In order to gain new insights on the mechanism by which spermidine is able to induce histone hypoacetylation in vivo, we monitored for the first time the in vitro effect of spermidine on H3 acetylation catalyzed by PCAF, a highly conserved histone acetyltrasferase (HAT). Interestingly, we found a specific inhibitory effect of spermidine in a broad range of concentration. However, we observed the opposite effect at very low spermidine concentrations, revealing an intriguing bimodal effect of spermidine on histone acetylation, depending exquisitely on its concentration. Moreover, we demonstrated for the first time the involvement of PCAF in the modification of spermidine to N8-acetylspermidine, which in turn might regulate the catalitic activity of PCAF itself.