Cortical areas of the juvenile rodent brain display a high degree of structural and functional plasticity, which disappears later in development. Coincident with the decline of plasticity 1) the hyaluronic acid-based extracellular matrix (ECM) of the brain, which stabilizes synapses and neuronal circuit is formed and 2) N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDARs) implied in synaptic plasticity switch from mainly GluN2B to GluN2A subunit-containing receptors. Here we tested the hypothesis that ECM influences the NMDAR subunit composition in dissociated neuronal cultures. Experimental removal of ECM using hyaluronidase induced an increase in surface expression of GluN2B. This was due to decreased endocytosis of surface GluNB-containing receptors. We further found a reduction in phosphorylation at Tyr1472, which negatively regulates their binding to the endocytotic AP2 complex. We propose that maturation of ECM could induce switch in NMDAR composition necessary for normal adult synaptic plasticity and that increased expression of GluN2B contributes to rejuvenation of plasticity after ECM removal in vivo.