Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.