Numerous studies have reported that hydroxyapatite nanoparticles (nano-HAPs) can inhibit the proliferation of a variety of tumor cells and this effect is different in different carcinoma cells. However, the effect of nano-HAPs on osteosarcoma cell proliferation has not been well understood thus far. In this study, we first showed that our synthesized nano-HAPs reduced cell viability and inhibited migration and invasion of OS-732 cells in a concentration-dependent manner. Using a BALB/c nude mouse tumor model, we demonstrated that nano-HAPs could effectively suppress tumor growth in vivo. We also performed RNA-seq analysis to investigate the underlying mechanism of these effects and discovered that treatment of OS-732 cells with nano-HAPs significantly downregulated the FAK/PI3K/Akt signaling pathway. Collectively, our study suggests that treatment with nano-HAPs can inhibit osteosarcoma cell growth, migration and invasion in vitro and suppress osteosarcoma in vivo.