OBJECTIVE: Neuropeptide Y (NPY) has been shown to have a prominent role in the control of bone formation through the regulation of osteoblast activity. We aimed to investigate the role of hypothalamus-derived NPY in bone metabolism. METHODS: Accordingly, adeno-associated virus (AAV)-mediated RNA interference (RNAi) was utilized to downregulate NPY gene expression in rats fed regular chow (RC) or a high-fat diet (HF). The serum concentrations of glucose, insulin, corticosterone, osteocalcin, insulin-like growth factor (IGF-1), triglycerides (TC), and cholesterol (TG) and fat mass and bone mineral density (BMD) were measured to assess the effect of NPY knockdown on basal and obesity-induced BMD. Forkhead transcription factor (FoxO1) and activating transcription factor 4 (ATF4) were measured to explore the molecular mechanism of the effect of dorsomedial nucleus (DMH) NPY knockdown on bone formation. RESULTS: Our results showed that DMH NPY knockdown enhanced basal and the obesity-induced decrease in BMD and osteocalcin and promoted the phosphorylation of FoxO1 and reduced the expression of ATF4. CONCLUSION: Our data suggest that DMH NPY knockdown can alter bone metabolism.