Purpose: Keratocyte to myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast and myofibroblast cell behavior. Methods: Primary rabbit corneal keratocytes, fibroblasts and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400, 800, 1200, 1600, 2000 and 4000 nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours and individual cell trajectories and migration rates were calculated. Results: Keratocytes, fibroblasts and myofibroblasts aligned and elongated to pitch sizes > 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes with a transition zone around the 800-1200 nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges > 1000 nm, but lacked directional guidance on submicron and nanoscale topographic features as well as on planar surfaces. Keratocytes remained essentially immobile. Conclusions: Corneal stromal cells elongated, aligned and migrated differentially guided by substratum topographic features. All cell types failed to respond to individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.