Calcyon is a single transmembrane endocytic protein that regulates clathrin assembly and clathrin-mediated endocytosis in the brain. Ultrastructural studies indicate that calcyon localizes to spines, but whether it regulates glutamate neurotransmission is not known. Here, we show that deletion of the calcyon gene in mice inhibits agonist-stimulated endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), without altering basal surface levels of the GluR1 or GluR2 subunits. Whole-cell patch-clamp studies of hippocampal neurons in culture and CA1 synapses in slices revealed that knockout (KO) of calcyon abolishes long-term synaptic depression (LTD), whereas mini-analysis in slices indicated basal transmission in the hippocampus is unaffected by the deletion. Further, transfection of green fluorescent protein-tagged calcyon rescued the ability of KO cultures to undergo LTD. In contrast, intracellular dialysis of a fusion protein containing the clathrin light-chain-binding domain of calcyon blocked the induction of LTD in wild-type hippocampal slices. Taken together, the present studies involving biochemical, immunological and electrophysiological analyses raise the possibility that calcyon plays a specialized role in regulating activity-dependent removal of synaptic AMPARs.