Cell adhesion is an important step in cell survival, and in the proliferation of anchorage-dependent cells, whose dimensions can be controlled by micro-patterning of the cell-adhesive extracellular matrix. To fabricate a micro-patterned fibronectin substrate with spacings ranging from 0.9 microm to 20 microm, we made a replica mold using e-beam lithography. The physiological behavior of human dermal fibroblasts (HDFs) on a substrate with a gradient of pattern spacings from 0.9 microm to 20 microm was evaluated after 4.5 hours and 2 days of culture. The number of proliferating cells on the fibronectin-patterned surface increased as the spacing between strip lines increased to 11 micron. However, the number of cells gradually decreased when the pattern spacing exceeded 11 microm. These findings demonstrate that the submicron-patterned topography of a substrate plays important roles in HDF survival and proliferation.