Gold nanoparticles have shown promising biological applications due to their unique properties. Understanding the interaction mechanisms between nanomaterials and biological cells is important for the control and manipulation of these interactions for biomedical applications. In the present study, we investigated the effects of gold nanoparticles on the differentiation of osteoblastic MC3T3-E1 cells and antimycin A-induced mitochondrial dysfunction. The results showed that gold nanoparticles (5, 10, and 20 nm) caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, and osteocalcin content in the cells (P < 0.05). Moreover, pretreatment with gold nanoparticles prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, cardiolipin peroxidation, and reactive oxygen species generation. Taken together, our study indicated that gold nanoparticles may improve the differentiation and have protective effects on mitochondrial dysfunction of osteoblastic cells.