<p>Osteosarcoma (OS) is characterized by high malignancy and profound metabolic reprogramming, yet the upstream regulators of its lipid metabolic adaptations remain largely elusive. Here, we report that RHBDL2 is significantly overexpressed in OS tissues, correlating with advanced clinical stage and poor patient prognosis. Mechanistically, multi-omics and structural analyses reveal that RHBDL2 functions as a non-proteolytic scaffold to stabilize the deubiquitinase USP3. This interaction is mediated by a compact hydrophobic core anchored by the Val245 residue of RHBDL2 and occurs independently of its protease activity. Stabilized USP3 subsequently prevents the proteasomal degradation of Palmitoyl-Protein Thioesterase 1 (PPT1) through deubiquitination. We further identify PPT1 as a metabolic rheostat that fuels OS malignancy by orchestrating FASN-dependent de novo lipogenesis, a requirement that can be partially bypassed by exogenous lipid supplementation. This RHBDL2-USP3-PPT1 axis promotes OS cell proliferation, migration, and epithelial-mesenchymal transition while suppressing apoptosis. Pharmacological screening identified Epigallocatechin gallate (EGCG) as a potent inhibitor that competitively disrupts the RHBDL2-USP3 interaction interface, thereby suppressing the downstream lipogenic program and inhibiting tumor growth and bone destruction in vivo. Collectively, our findings delineate a novel signaling cascade linking post-translational protein stabilization to metabolic adaptation, highlighting the RHBDL2-USP3 structural interface as a promising therapeutic vulnerability in osteosarcoma.</p>