Myotonic dystrophy type 1 (DM1) is a severe multisystemic condition. The impairment of the central nervous system (CNS) is demonstrated by cognitive and attention deficits, executive dysfunction, prevalent hypersomnia, behavioral changes, as well as intellectual disability in the most severe cases. DM1 is caused by the abnormal expansion of a non-coding trinucleotide CTG repeat. Expanded CUG transcripts accumulate in toxic RNA aggregates (or foci) in the cell nucleus, which perturb primarily the regulation of alternative splicing. Important gaps still exist in our understanding of the disease mechanisms in the brain: we do not know the cell types and the molecular pathways most predominantly affected, and how they contribute to the onset of the debilitating neurological manifestations of DM1. Using a transgenic mouse model of DM1 we found preferential accumulation of toxic RNA foci and missplicing in cortical astrocytes, relative to neurons, pointing to glia cell pathology. We used our DM1 mice as a source of primary neurons and astrocytes to resolve cell type-specific disease mechanisms by RNA sequencing of homogenous cell cultures. DM1 mouse astrocytes confirmed greater RNA foci accumulation and showed critical missplicing of transcripts that regulate cell adhesion, cytoskeleton dynamics and cell morphogenesis. Astrocyte spliceopathy translated into defective cell adhesion, reduced spreading and erratic migration in culture, as well as decreased astrocyte ramification and aberrant reorientation in DM1 mouse brains. We confirmed the abnormal splicing of relevant transcripts in brain tissue from DM1 patients, and the defective spreading of human glia cells expressing toxic CUG RNA in culture. In conclusion, we have shown the CTG repeat expansion has a deleterious impact on glia cell biology, which may impair the glial-neuronal crosstalk and synaptic function in DM1 brains, contributing to cognitive and behavioural deficits. Our results provide new insight into the cellular and molecular mechanisms of DM1 brain disease.