Culturing cells on planar substrate in vitro is a conventional cell biology method. However, each type of physiological tissues has its specific three-dimensional micro-structure, which provides various micro environment to regulate such biological processes as cell proliferation and differentiation. To date, a growing body of researches on the impacts of substrate micro-topography on cellular responses has been documented in the literature. It is found that micro-topograhical substrate can manipulate cell spreading, migrating, orientating, cytoskeleton remodeling, and stem cell differentiation, which are crucial to ex vivo tissue construction and surface modification of medical implanting materials. This review discusses the recent progresses of the effects of substrate micro-topography on cellular responses and the underlying mechanisms of mechano-biological coupling.