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Cells are known to sense the topographic features of the substrate and align along the direction of the surface pattern, and this is believed to be an important aspect in the formation and regeneration of anisotropic biological tissues. In this study, a unique and anisotropic stepped pattern was produced on single crystals of α-Ti with the h.c.p. lattice by plastic deformation in compression to demonstrate the effect of the pattern on cell behavior. Because the Schmid factor for the operative slip system of prismatic (100)[110] was set to be 0.5, the slip traces with an acute angle of 45° appeared on the surface. A smooth substrate without plastic deformation was used as a control. MC3T3-E1 osteoblastic cells were cultured on the substrate for 24 h, followed by observation of the morphology and alignment of the cells by Giemsa staining. On stepped substrates, cells aligned along the slip traces, and the filopodia of the aligned cells were found to extend parallel to the slip traces. The slip traces induced by plastic deformation of a single crystal was successfully proven to be a potent substrate to control the alignment of cells.