Neuronal communication relies on neurotransmitter release from synaptic vesicles (SVs), whose dynamics are controlled by Ca2+-dependent pathways, as many thoroughly studied phosphorylation cascades. However, little is known about other post-translational modifications, as ubiquitination. To address this, we analysed resting and stimulated synaptosomes (isolated synapses) by quantitative mass spectrometry. We identified more than 5,000 ubiquitination sites on ~2,000 proteins, the majority of which participate in the SV recycling processes. Several proteins showed significant changes in ubiquitination in response to Ca2+ influx, with the most pronounced changes in CaMKIIα and the clathrin adaptor protein AP180. To validate this finding, we generated a CaMKIIα mutant lacking the ubiquitination target (K291), and analyse it in both neurons and non-neuronal cells. K291 ubiquitination influences CaMKIIα activity and synaptic function, by modulating its autophosphorylation at a functionally important site (T286). We suggest that activity-dependent ubiquitination is an important regulator of synaptic function.