Nanomaterials are attractive for use in biological systems due to their ability to control the microenvironment of cells. Additionally, nanofibers can mimic fibrous characteristics of natural tissues. This study was conducted to assess astrocyte activity and infiltration behavior on Spirulina extract-embedded polycaprolactone (SP-PCL) nanofiber. Astrocytes moved along with the nanofiber, and developed an elongated and stellate shape, which is similar to those in the natural neural tissue. In addition, the expression of GFAP, a biomarker representing the activation of astrocytes, was gradually up-regulated with the increase of the concentration of Spirulina extract, indicating that Spirulina extract can control astrocyte activation. Overall, the results presented herein indicate that SP-PCL nanofiber could be used in astrocyte tissue engineering for neuronal regeneration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 3467–3473, 2013.