Abstract Prion diseases are neurodegenerative disorders with long asymptomatic incubation periods, followed by a rapid progression of cognitive and functional decline culminating in death. The complexity of intercellular interactions in the brain is challenging to unravel and the basis of disease pathobiology remains poorly understood. In this study, we employed single cell RNA sequencing (scRNAseq) to produce an atlas of 147,536 single cell transcriptomes from cortex and hippocampus of mice infected with prions and showing clinical signs. We identified transcriptionally distinct populations and sub-populations of all the major brain cell-types. Disease-related transcription was highly specific to not only overarching cell-types, but also to sub-populations of glia and neurons. Most striking was an apparent decrease in relative frequency of astrocytes expressing genes that are required for brain homeostasis such as lipid synthesis, glutamate clearance, synaptic modulation and regulation of blood flow. Additionally, we described a spectrum of microglial activation states that suggest delineation of phagocytic and neuroinflammatory functions in different cell subsets. Differential responses of immature and mature neuron populations were also observed, alongside abnormal hippocampal neurogenesis. Our scRNAseq library provides a new layer of knowledge on single cell gene expression in prion disease, and is a basis for a more detailed understanding of cellular interplay that leads to neurodegeneration.