Stimulation of metabotropic glutamate receptors (mGluRs) belonging to group I has been found to reduce N-methyl- d-aspartate (NMDA) receptor function in terms of both intracellular calcium concentration ([Ca2+]i) rise and neurotoxicity in cultured cerebellar granule cells. In the present study, we investigated whether the mGluR-elicited modulation of glutamate responses might rely on the heteromeric composition of NMDA receptor channel. NMDA receptors consist of two distinct groups of subunits: NR1, that is ubiquitously in the receptor complexes; and NR2A–D, that differentiate and potentiate NMDA receptor responses by assembling with NR1. Among NR2 subunits, only NR2A and NR2C mRNAs and relative proteins are detected in cerebellar granule cells at 10 days in vitro. To dissect the involvement of the two different subunits in making the NMDA receptor channel sensitive to modulation by group I mGluR agonists, expression of the NR2C subunit was prevented by treating the cells with specific antisense oligodeoxynucleotide (ODN). The capability of the mGluR agonists, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD, 100 μm) or 3 hydroxyphenylglycine (3HPG, 100 μm), and the protein kinase C (PKC) activator, 4β-phorbol-12,13-dibutyrate (PDBu, 1 μm), to inhibit the function of resultant NMDA receptors was then evaluated. We found that depletion of the NR2C subunit abolished the inhibitory effect of group I mGluR stimulation on glutamate-induced [Ca2+]i rise and neurotoxicity. The antisense ODN treatment also prevented the inhibitory effect of PDBu on glutamate responses. Conversely, in NR2C-lacking neurons, both group I mGluRs and PKC stimulation enhanced NMDA receptor-mediated effects. The present findings indicate that the capability of PKC-associated mGluRs to modulate native NMDA receptor function relies on the heteromeric configuration of the receptor–channel complex. Particularly, expression of the NR2C subunit is required to make the NMDA receptor sensitive to inhibitory modulation by mGluRs or PKC activation.