The synapse contains densely localized and interacting proteins that enable it to adapt to changing inputs. We describe a Ca2+-sensitive protein complex involved in the regulation of AMPA receptor synaptic plasticity. The complex is comprised of MUPPI, a multi-PDZ domain-containing protein; SynGAP, a synaptic GTPase-activating protein; and the Ca2+/calmodulin-dependent kinase CaMKII. In synapses of hippocampal neurons, SynGAP and CaMKII are brought together by direct physical interaction with the PDZ domains of MUPP1, and in this complex, SynGAP is phosphorylated. Ca2+CaM binding to CaMKII dissociates it from the MUPP1 complex, and Ca2+ entering via the NMDAR drives the dephosphorylation of SynGAP. Specific peptide-induced SynGAP dissociation from the MUPP1-CaMKII complex results in SynGAP dephosphorylation accompanied by P38 MAPK inactivation, potentiation of synaptic AMPA responses, and an increase in the number of AMPAR-containing clusters in hippocampal neuron synapses. siRNA-mediated SynGAP knockdown confirmed these results. These data implicate SynGAP in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking.