Protein acetylation affects synaptic plasticity and memory, but its effects on synapse composition have not been addressed. We found that protein acetylation promotes the dendritic clustering of the excitatory postsynaptic scaffold protein PSD95 in hippocampal neurons, without affecting the total levels of this protein. Cortactin, an F-actin-binding protein enriched in dendritic spines, is a substrate for acetylation and has a role in spine morphogenesis. Recent studies showed that cortactin acetylation changes its ability to bind F-actin and regulates cellular motility, but the function of cortactin acetylation in neuronal cells is so far unknown. We tested whether acetylation of cortactin influences its morphogenic function by overexpressing wild-type cortactin, or the mimetic mutants for acetylated or deacetylated cortactin, in hippocampal neurons, and found that cortactin acetylation has an impact on PSD95 clustering, independent from its function as actin dynamics regulator. Moreover, acetylated cortactin can rescue the reduction in PSD95 clustering mediated by knockdown of cortactin. We also found that acetylation of cortactin is correlated with decreased cortactin interaction with p140Cap and Shank1, and with lower cortactin phosphorylation at tyrosine 421. The neurotrophin BDNF promoted the acetylation of cortactin in hippocampal neurons, suggesting that BDNF may regulate excitatory synapses and PSD95 dendritic clustering at least in part by changing the acetylation level of cortactin. Our findings unravel an unsuspected role for cortactin acetylation in the regulation of PSD95 dendritic clustering, which may work in concert with cortactin's role in spine development.