The topography of electrospun fiber scaffolds modifies astrocytes toward -like morphologies and behaviors. However, little is known about how electrospun fiber diameter influences astrocyte behavior. In this work, aligned fibers with two distinct nanoscale fiber diameters (808 and 386 nm) were prepared, and the astrocyte response was measured over time. Astrocytes on the large diameter fibers showed significantly increased elongation as early as 2 h after seeding and remained significantly more elongated for up to 4 days compared to those on small diameter fibers. Astrocytes extending along larger diameter fibers were better equipped to support long neurite outgrowth from dorsal root ganglia neurons, and neurite outgrowth along these astrocytes was less branched than outgrowth along astrocytes cultured on small diameter fibers. The differences in astrocyte shape observed on the small or large diameter fibers did not translate into differences in GLT-1, GFAP, or GLAST protein expression. Thus, different fiber diameters were unable to influence astrocyte protein expression uniquely. Nevertheless, astrocytes cultured in either small or large fibers significantly increased their expression of GLT-1 compared to astrocytes cultured on nonfiber (film) controls. Fibrous-induced increases in astrocyte GLT-1 expression protected astrocyte/neuron cocultures from toxicity generated by high extracellular glutamate. Alternatively, astrocytes/neurons cultured on films were less able to protect these cells from culture conditions consisting of high glutamate levels. Biomaterials, such as the fibrous materials presented here, may help stimulate astrocytes to increase GLT-1 expression and uptake more glutamate, since astrocytes are less likely to uptake glutamate in neurodegenerative pathologies or following central nervous system injury.