Adenine is an endogenous purine base that plays essential roles in cellular energy metabolism and nucleotide synthesis. In this study, we demonstrate that adenine promotes osteoblast differentiation through activation of the AMPK–CREB signaling pathway. In C2C12 cells, adenine treatment increased AMPK and phosphorylated AMPK levels, accompanied by enhanced CREB phosphorylation and subsequent upregulation of osteogenic markers, including RUNX2, ALP, OPN, and collagen. Pharmacological inhibition of AMPK using Compound C suppressed CREB phosphorylation and significantly reduced the expression of RUNX2, ALP, and collagen, confirming that adenine-induced osteogenic signaling is AMPK-dependent. These findings indicate that AMPK acts as a critical upstream regulator in adenine-mediated osteogenesis. Furthermore, adenine enhanced both early and late markers of osteoblast differentiation, suggesting its involvement across multiple stages of osteogenesis. In vivo, dietary supplementation with 0.02% adenine for 10 weeks significantly improved tibial bone density and thickness in ovariectomized (OVX) mice, as demonstrated by H&E staining. Histological analysis showed restoration of trabecular architecture and increased cortical thickness compared with OVX controls. Notably, bone parameters in adenine-treated OVX mice were comparable to those in the sham-operated group, indicating a protective effect against estrogen deficiency–induced bone loss.