Surface roughness has been considered as an important influencing factor for cell response. The aim of this study was to find out whether MC3T3-E1 cells, a mouse osteoblast-like cell line, can sense the amplitudes of surface topography of titanium alloy (Ti6Al4V), and if surface-dependent cell morphology would be presented on the substrata with varied roughness. A series of polished samples (Ra: 0.30~1.80 μm) were prepared to produce macroscopically parallel grooves using different grades of silicon carbide sandpaper (#100, #320, #600, #1000 and #2000). The experimental results indicated that the behavior and morphology of cells largely depended on the substrata where they were cultured. More efficient proliferation of MC3T3-E1 cells was shown on the surfaces with Ra of 0.50~1.00 μm, with respect to either the rougher or the smoother specimens. Furthermore, MC3T3-E1 cells seeded on the Ti6Al4V surfaces within this narrow range responded to the increasing surface roughness with increased proliferation. Contact guidance of cells could be observed on the rougher specimens (Ra: 0.80~1.00 μm), whereas more random orientations were exhibited for the adsorbed cells on the smoother surfaces (Ra: 0.50~0.60 μm).