The contribution of fiber alignment of scaffold on cellular mechanisms was evaluated by a comparative study of two different cells sourced from cornea. Electrospun scaf-folds with similar composition and comparable fiber size were fabricated into randomly oriented and aligned scaffolds, which bear paralleled degradation of gelatin. Tensile test of wet scaffolds indicated that fiber alignment could influence its mechanical properties. Due to the unidirectional fiber orientation, aligned scaffold exhibited higher tensile modulus, higher break strength, and lower elongation at break than randomly oriented scaffold. The effect of fiber alignment on cells behavior was evaluated by cell morphology, specific protein expression, adhesion, and proliferation. Different corneal cells responded uniquely to fiber alignment of scaffold, keratocytes interacting more favorably on alignment scaffold and corneal epithelial cells more favorably on randomly oriented scaffold. These results confirmed that fiber alignment of scaffold would be benefit for cell proliferation if its contact guidance coincided with the cell shape and cytoskeletal tension. This finding is important to envisage an advanced composite scaffold that incorporates randomly oriented and aligned fibers for the growth and control of different cell types required for the successful development of corneal grafts by tissue engineering. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.