The small ubiquitin-like modifier protein (SUMO) regulates transcriptional activity and the translocation of proteins across the nuclear membrane. The identification of SUMO substrates outside the nucleus is progressing but little is yet known about the wider cellular role of protein SUMOylation. Here we report that in rat hippocampal neurons multiple SUMOylation targets are present at synapses and we show that the kainate receptor subunit GluR6 is a SUMO substrate. SUMOylation of GluR6 regulates endocytosis of the kainate receptor and modifies synaptic transmission. GluR6 exhibits low levels of SUMOylation under resting conditions and is rapidly SUMOylated in response to a kainate but not an N-methyl-D-aspartate (NMDA) treatment. Reducing GluR6 SUMOylation using the SUMO-specific isopeptidase SENP-1 prevents kainate-evoked endocytosis of the kainate receptor. Furthermore, a mutated non-SUMOylatable form of GluR6 is not endocytosed in response to kainate in COS-7 cells. Consistent with this, electrophysiological recordings in hippocampal slices demonstrate that kainate-receptor-mediated excitatory postsynaptic currents are decreased by SUMOylation and enhanced by deSUMOylation. These data reveal a previously unsuspected role for SUMO in the regulation of synaptic function.