Multiple sclerosis (MS) is chronic disease of the central nervous system characterized by demyelinated lesions in both grey and white matter. While white matter lesions are typically associated with inflammatory demyelination, grey matter lesions (GML) often indicate ongoing neurodegenerative processes. GML are linked to clinical disability, though non-demyelinated areas in grey matter also exhibit underlying neuropathology. In grey matter, the extracellular matrix (ECM) plays a vital role in maintaining neuronal health, with perineuronal nets (PNNs) functioning as barriers, regulating synaptic stability and limiting postsynaptic receptor mobility. Microglia impact ECM composition by secreting ECM-degrading matrix metalloproteinases (MMPs), while the ECM, in turn, affects microglia behaviour. In this study, we examined differences in non-cell-associated ECM composition between human control and MS tissue and investigate how this ECM influences microglia. Label-free quantitative mass spectrometry of decellularized control grey matter (dCGM) and normal appearing grey matter (dNAGM) tissue slices revealed differentially abundant ECM proteins, and several ECM proteins uniquely present in either dNAGM or dCGM. The altered ECM composition of dNAGM involved changes in PNN, synaptic and basement membrane components, potentially impacting synaptic and blood-brain barrier function. The ECM composition of decellularized GML (dGML) and surrounding perilesional grey matter (dPLGM) appeared relatively similar. When decellularized human grey matter brain tissue was recellularized with primary microglia, half of the microglia population lost IBA1 expression on dNAGM and dGML, while a portion of the remaining IBA1+ microglia expressed the proinflammatory marker iNOS . These findings suggest changes in non-cell-associated ECM composition in the absence of demyelination, potentially influencing microglial behaviour and contributing to MS pathology.