Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates.