Synaptic transmission, the linchpin in neuronal circuits, is exquisitely controlled by protein synthesis and possibly by protein degradation via the ubiquitin proteasome system (UPS). In addition to altering protein life-time, ubiquitination can also alter protein function. Recent data suggests a role for the UPS in regulation of presynaptic function, but it remains unclear whether ubiquitination at the synapse serves mainly as degradative signal. We used two distinct classes of UPS blockers to determine the effect of UPS inhibition on synaptic function in cultured hippocampal neurons. We find that proteasome inhibitors as well as E1 ubiquitin-activating enzyme inhibitors trigger, within minutes, a several-fold increase in the frequency of excitatory and inhibitory miniature postsynaptic currents. Taken together with the finding that protein synthesis inhibitors do not block the UPS inhibition-mediated increase in mini frequency, these data suggest that ubiquitination at the presynaptic terminal not only regulates the half-life, but also the functional state of proteins. We conclude that, beyond the classic degradatory pathway, the UPS serves as an important and powerful regulator of synaptic activity in the presynaptic terminal by modulating protein function.