Objective To quantify the micro-geometric and configuration of micropatterned substrate and analyze its effects on proliferation, differentiation, and migration of bone marrow mesenchymal stem cells (BM MSCs). Method Micropatterned substrates were designed and fabricated to control cells' spreading shape and area. Data of proliferation, differentiation and migration of rat BM MSCs on various patterned substrates were compared. Result Proliferation of BM MSCs was inhibited when their spreading width was narrowed. Osteoblastic differentiation was regulated distinctively in different spreading shape and area. Cell migration was more active when their spreading area was reduced. Dexamethasone was found to play an important role in down-regulating cell proliferation and migration on patterned substrate. Conclusions Cell spreading shape and area is important factors for regulating proliferation, differentiation, and migration of BM MSCs.