Astrocytes produce extracellular matrix (ECM) glycoproteins contributing to the blood-brain barrier and regulating the immune response in the central nervous system (CNS). The aim of this study was to investigate the impact of astrocyte depletion upon the clinical outcome and the composition of ECM glycoproteins in a virus-induced animal model of demyelination. Glial fibrillary acidic protein (GFAP)-thymidine-kinase transgenic SJL (GFAP-knockout) and wildtype mice were infected with Theiler's murine encephalomyelitis virus (TMEV). Astrocyte depletion was induced during the progressive, demyelinating disease phase by ganciclovir administration once daily between 56 and 77 days post infection (dpi). At 77 dpi GFAP-knockout mice showed a significant deterioration of clinical signs associated with a reduction of azan and picrosirius red stained ECM-molecules in the thoracic spinal cord. Basement-membrane-associated ECM-molecules including laminin, entactin/nidogen-1 and Kir4.1 as well as non-basement membrane-associated ECM-molecules like collagen I, decorin, tenascin-R and CD44 were significantly reduced in the spinal cord of GFAP-knockout mice. The reduction of the investigated ECM-molecules demonstrates that astrocytes play a key role in the production of ECM-molecules. The present findings indicate that the detected loss of Kir4.1 and CD44 as well as the disruption of the integrity of perineuronal nets led to the deterioration of clinical signs in GFAP-knockout mice.