Microgrooved surfaces have been used extensively to influence cell contact guidance. Guiding cell growth, extracellular matrix deposition, and mineralization is important for bone implant longevity. In this study, we investigated the osteoblast response to microgrooved metallic surfaces in serum-supplemented medium. Groove spacing was comparable with the spread osteoblast size. Focal adhesions were observed to confine to the intervening ridge/groove boundaries. Osteoblasts bridged over the grooves and were unable to conform to the concave shape of the underlying grooves. Microgrooved surfaces induced higher osteoblast proliferation and metabolic activity after 14 days in osteogenic medium compared with as-received surfaces, resulting in higher mineralization and alignment of cell-secreted collagen after 28 days. To establish whether preferential cell attachment at the ridge/groove boundaries was influenced by the adhesion proteins contained in the serum-supplemented media, fluorescently labeled fibronectin was adsorbed onto the microgrooved substrates at low concentrations, mimicking the concentrations found in blood serum. Fibronectin was found to selectively adsorb onto the ridge/groove boundaries, the osteoblast focal adhesion sites, suggesting that protein adsorption may have influenced the cell attachment pattern.