We tested the hypothesis that astrocytes grown in a suitable three-dimensional (3D) cell culture system exhibit morphological and biochemical features of in vivo astrocytes that are otherwise lost upon transfer from the in vivo to a two-dimensional (2D) culture environment. First, we report development of a novel bioactively coated nanofiber-based 3D culture system (Bioactive3D) that supports cultures of primary mouse astrocytes. Second, we show that Bioactive3D culture system maintains the in vivo-like morphological complexity of cultured cells, allows movement of astrocyte filopodia in a way that resembles the in vivo situation, and also minimizes the cellular stress, an inherent feature of standard 2D cell culture systems. Third, we demonstrate that the expression of gap junctions is reduced in astrocytes cultured in a 3D system that supports well-organized cell-cell communication, in contrast to the enforced planar tiling of cells in a standard 2D system. Finally, we show that astrocytes cultured in the Bioactive3D system do not show the undesired baseline activation but are fully responsive to activation-inducing stimuli. Thus, astrocytes cultured in the Bioactive3D appear to more closely resemble astrocytes in vivo and represent a superior in vitro system for assessing (patho)physiological and pharmacological responses of these cells and potentially also in co-cultures of astrocytes and other cell types.