Background <p>Lipid metabolism disorders in osteoblasts may lead to osteoporosis. CUB domain-containing protein 1 (CDCP1) is associated with various intracellular signaling pathways. We investigated how CDCP1 regulates lipid metabolism and osteoblast function.</p> Methods <p>This study utilized gene overexpression (via lentivirus) and loss-of-function (CRISPR/Cas9-mediated knockout) techniques to investigate the involvement of CDCP1 in lipid metabolism and osteogenesis. Transcriptomic and metabolomic analyses were performed to examine the mechanism of action of CDCP1. Furthermore, proximity ligation assays, GST pull-down, and molecular docking were employed to identify the interaction between CDCP1 and AMP-activated protein kinase (AMPK).</p> Results <p>CDCP1 alleviated bone loss in mice. In vitro, CDCP1 promoted the phosphorylation of AMPK. Phosphorylated AMPK can enhance the activity of carnitine palmitoyltransferase, leading to increased fatty acid oxidation and promoting osteogenesis. Mechanistically, CDCP1 prevents the formation of the autoinhibitory conformation of the autoinhibitory domain by binding to the α3 helix, thereby protecting AMPK phosphorylation from self-inhibition.</p> Conclusions <p>Our research revealed a new molecular mechanism linking CDCP1 and allosteric control of AMPK. These findings reveal for the first time the mechanism by which CDCP1 affects osteogenesis through lipid metabolism regulation, suggesting its potential as a therapeutic target for osteoporosis.</p> Graphical abstract <p></p>