Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood‐brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S‐100 immunohistochemical staining patterns in the hippocampus of wild‐type and mutant mice suggested a normal abundance of astrocytes in GFAP‐negative mice, however, in contrast to wild‐types, GFAP‐negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up‐regulation of other intermediate filament proteins, such as vimentin. The GFAP‐negative mice displayed post‐traumatic reactive gliosis, which suggests that GFAP up‐regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.