The role of topographical pattern on cell behavior regulation has been recognized but has not been elucidated yet. Here, using soft lithography techniques to allow mass-production of microgrooved substrates with varying dimensions at mesoscopic scale (from single-cell to multicellular scale), we identified the spatial distribution of cancer cells in response to topographical pattern due to contact guidance. We observed that increasing depth and groove width is accompanied by more obvious preferential distribution on ridges. Determination of cell density and migrate velocity signatures reveals that the preferential distribution on ridges is caused by cell upward migration. Then, based on the investigation of influences of soluble factors concentration and quantification of cell-substrate adhesion, the cell upward motility was considered to be governed by cell-substrate adhesion caused by contact guidance due to different constraints. Interestingly, in response to the topological feature dimensions, the upward migration of cells is driven by alternative mechanisms based on FAs (focal adhesions) constraints (single-cell scale) and entropic maximization (multicellular scale). Understanding the mechanism of contact guidance on mesoscopic scale provides basic support for the study of cell alignment and migration, thereby aiding in disease progression prediction, especially for cancer metastasis.