Astrocytes play an active role in the brain, including structural and metabolic support of neurons as well as gliotransmitter release. For the interaction of astrocytes with the surrounding extracellular matrix and neighboring cells the formation of localized contacts is required. One common adaptor of cell-matrix and cell-cell adhesions is the protein vinculin which structurally and functionally links the cell adhesion receptors to the actin cytoskeleton and which has been implicated in regulation of many cellular processes by in vitro experiments. To analyze the role of vinculin in astrocytes in vivo, we generated a transgenic mouse model allowing inducible astrocyte-specific inactivation of vinculin by using Cre-loxP technology. Genotyping of single astrocytes isolated from different brain regions demonstrated successful recombination of the floxed vinculin gene in vivo. Quantitative PCR showed a decrease of vinculin mRNA, while quantification of fluorescence images revealed the absence of vinculin in astrocytes in the knockout animals. To establish the impact of vinculin deficiency, expression and localization of proteins involved in astroglial function were analyzed by immunohistochemistry. Numerous proteins were not affected by the loss of vinculin including the gap junction protein connexin43. Also no indication for reactive gliosis could be detected in the knockout animals. In contrast to in vitro studies, our surprising results suggest that vinculin is much less important for proper astrocyte function in mice in vivo and add a note of caution on the extrapolation of in vitro data to in vivo function.