To investigate the cellular response to designed topography in vitro, we studied the adhesion, proliferation, osteogenic differentiation, and calcification of mouse preosteoblasts (MC3T3-E1) cultured on titanium (Ti) surfaces with periodic micrometer scale grooves containing nanometer scale ripples in the vertical direction fabricated by single-shot, femtosecond laser irradiation (fsTi). The surface composition and chemical state of fsTi were almost the same as those of mirror-polished Ti without femtosecond laser irradiation (mTi). Cells cultured on fsTi were highly aligned, whereas the cell proliferation rate on fsTi was less than that on mTi. Higher gene expressions of Spp1 and Bglap1 were detected in cells cultured on fsTi than those on mTi, indicating that the periodic micro/nanometer scale grooves topography promoted osteogenic differentiation and calcification. This initial activation of osteoinduction on fsTi generated calcified deposits that were thicker and larger than those on mTi and hence, osteoconductivity was promoted on fsTi. Our findings indicate that femtosecond laser irradiation is a technique with potential for controlling biomaterial-cell interfaces and, in particular, the promotion of osseointegration of Ti. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3456-3464, 2017.