Astrocytes are rapidly gaining ground as important regulators and potential therapeutic targets in neurodegeneration. Here, we used the cuprizone model of multiple sclerosis to explore the phenotypic signature of astrocytes in the cortex of mice during early demyelination at week 3 (CPZ3), a timepoint of initial demyelination and glia cell activation. Confocal imaging combined with detailed cell-morphometric analysis of cortical astrocytes after 3-dimensional cell reconstruction revealed the morphological phenotype of astrocytes in early demyelination, characterized by thick and long processes. To define the molecular phenotype of these astrocytes, we performed single-cell RNA sequencing in isolated cortical tissues from healthy and CPZ3-disease brains. Differential gene analysis revealed a significant number of regulated genes with enrichment of genes typical of pan-reactive astrogliosis (e.g. Gfap, Vim, Osmr), while markers widely associated with neurotoxic or protective astrocytes appeared mostly unaffected. We further report two specific clusters, present in healthy brains, but highly regulated in disease. These clusters were characterized by regulation of genes involved in neuroinflammation and neuroprotection, and one was further characterized by robust upregulation of typical reactive astrocyte markers, recently attributed to distinct astrocyte subpopulations in a mouse model for Alzheimer’s Disease, and downregulation of markers involved in physiological astrocyte functions. Our data provides a detailed phenotypic and molecular characterization of grey matter astrocyte responses in early experimental demyelination that might provide a basis for investigation and understanding of astrocyte mechanisms involved in pathology and for the design of strategies aimed at regulating astrocyte responses for the treatment of neurodegenerative diseases.