The nanoscale anisotropic patterns of bioactive ligands in the extracellular matrix regulate cell adhesion behaviors. However, the mechanisms of such regulation remain unclear. Here, RGD-bearing gold nanorods (AuNRs) are conjugated with different aspect ratios (ARs, from 1 to 7) on cell culture substrates to decouple the effect of nanoscale anisotropic presentation of cell adhesive RGD peptides on cell adhesion. Compared with AuNRs with small ARs, AuNRs with large ARs significantly promote cell spreading, the alignment of the basal cytoskeletal structure, and nanopodia attachment. Furthermore, both -β3 and -β1 class integrins are recruited to AuNRs with large ARs, thereby promoting the development of focal adhesion toward fibrillar adhesion, whereas the recruitment of diverse integrins and the development of cell adhesion structures are hindered by small ARs AuNRs. The anisotropic presentation of ligands by large AR AuNRs better activates mechanotransduction signaling molecules. These findings are confirmed both in vitro and in vivo. Hence the enhanced mechanotransduction promotes osteogenic differentiation in stem cells. These findings demonstrate the potential use of well-controlled synthetic nanoplatforms to unravel the fundamental mechanisms of cell adhesion and associated signaling at the molecular level and to provide valuable guidance for the rational design of biomaterials with tailored bioactive functions.