Extracellular matrix (ECM) plays a very important role in regulating cell function and fate. It is highly desirable to fabricate biomimetic models to investigate the role of ECM in stem cell differentiation. In this study, arginine–glycine–aspartate (RGD)-modified gold nanoparticles (Au NPs) with tunable surface ligand density were prepared to mimic the ECM microenvironment. Their effect on osteogenic and adipogenic differentiation of human mesenchymal stem cells (MSCs) was investigated. The biomimetic Au NPs were taken up by MSCs in a ligand density-dependent manner. The biomimetic NPs with a high RGD density had an inhibitive effect on the alkaline phosphatase (ALP) activity, calcium deposition, and osteogenic marker gene expression of MSCs. Their effect on oil droplet formation and adipogenic marker gene expression was negative when RGD density was low, while their effect was promotive when RGD density was high. The biomimetic Au NPs regulated the osteogenic and adipogenic differentiation of MSCs mainly through affecting the focal adhesion and cytoskeleton. This study highlights the roles of biomimetic NPs on stem cell differentiation that could provide a meaningful strategy in fabricating functional biomaterials for tissue engineering and biomedical applications.