Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.