Understanding the interaction mechanisms between nanomaterials and biological cells is important for the control and manipulation of these interactions for biomedical applications. In this study, we investigated the cellular effects of gold nanoparticles (AuNPs) on the differentiation of mesenchymal stem cells (MSCs) and the associated molecular mechanisms. The results showed that AuNPs promoted the differentiation of MSCs toward osteoblast cells over adipocyte cells by inducing an enhanced osteogenic transcriptional profile and an attenuated adipogenic transcriptional profile. AuNPs exerted the effects by interacting with the cell membrane and binding with proteins in the cytoplasm, causing mechanical stress on the MSCs to activate p38 mitogen-activated protein kinase pathway (MAPK) signaling pathway, which regulates the expression of relevant genes to induce osteogenic differentiation and inhibit adipogenic differentiation.