Cellular Prion Protein (PrP(C)) is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrP(SC)) induces transmissible spongiform encephalopathies (TSEs). In contrast, PrP(C) has a number of physiological functions in several neural processes. Several lines of evidence implicate PrP(C) in synaptic transmission and neuroprotection, since its absence results in an increase in neuronal excitability, and enhanced excitotoxicity in vitro and in vivo. Further, PrP(C) has been implicated in the inhibition of NMDA-mediated neurotransmission, and Prnp knockout mice show enhanced neuronal death in response to NMDA and Kainate (KA). In the present study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the JNK3 pathway, since Prnp(o/o)Jnk3(o/o) mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrP(C)-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrP(C) with PSD-95 and GluR6/7. Indeed, GluR6-PSD-95 interaction after KA injections was favoured by the absence of PrP(C). Lastly, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (DNQX) and the GluR6 antagonist (NS-102). We conclude that the protection afforded by PrP(C) against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.