Introduction: Astrocytes are the most abundant cells in the mammalian brain and play important roles in regulating neuronal signaling, protecting neurons, and determining the fate of neural precursors. Astrocytes are one of the best candidates for cell therapy. Three-dimensional (3D) scaffolds are a synthetic matrix which provided a biocompatible, biodegradable, and non-toxic 3D environment for a variety of cells. In the present study, we isolated astrocytes from rat neocortex and assessed the proliferation rate of astrocytes in 3D dimensional environment. Materials and Methods: Astrocytes isolated from adult rat neocortex. The isolated cells was cultured in DMEM/F12 medium supplement with 10% FBS. Astrocytes were confirmed by expression of GFAP (glial fibrillary acidic protein), assessed by immunocytochemistry. After the 3th passage, the astrocytes were cultured in 3D and two - dimensional (2D) cultures and MTS assay was used to determined cell proliferation. Results: The expression of GFAP marker was confirmed by immunocytochemistry assay. Our results showed that surface plating of astrocytes with 0.15% Pura Matrix showed significantly greater cell proliferation in comparison with 2D culture. Conclusion: These findings indicate the potential of the astrocytes which was cultured in 3D for using in cell transplantation.