At glutamatergic brain synapses, scaffolding proteins regulate receptor location and function. The targeting and organization of scaffolding proteins in the postsynaptic density (PSD) is poorly understood. A core protein of the glutamatergic receptor postsynaptic scaffold complex, GKAP, interacts with DLC2, a protein associated with molecular motors. In the present study, we combined BRET imaging, immuno-staining and electrophysiological recording to assess the role of GKAP-DLC2 interaction in the functional organization of the glutamatergic synapse. We found that GKAP-DLC2 interaction in dendritic spine stabilizes scaffolding protein expression at the PSD and enhances synaptic NMDA receptor activity. Moreover, the GKAP-DLC2 functional interaction is favored by sustained synaptic activity. These data provide a novel regulatory pathway of synaptic transmission that depends on activity-induced remodeling of the postsynaptic scaffold protein complex.