Understanding the interfacial structure between nanomaterials and lipoproteins is crucial for gaining profound insights into how nanomaterials impact lipoprotein structure and interfere with lipid metabolism. In this study, we employed graphene oxide (GO) nanosheets (NSs) with precisely controlled surface hydrophilicity as model nanomaterials to investigate the influence of surface properties on lipoprotein corona formation. Our findings reveal that hydrophobic GOs exhibit a greater affinity for binding with low-density lipoprotein (LDL). To explore the interaction between GOs and LDL at the interface, we employed advanced techniques such as X-ray reflectivity, circular dichroism, and molecular simulation. Specifically, hydrophobic GOs showed a preference for associating with the lipid components of LDL, while hydrophilic ones tended to bind with apolipoproteins. Furthermore, our research demonstrated that these GOs distinctly modulate various lipid metabolism pathways, including LDL recognition, uptake, hydrolysis, efflux, and lipid droplet formation. This comprehensive investigation underscores the significance of structural analysis at the nano-biomolecule interface and emphasizes the critical role of nanomaterial’s surface properties in mediating cellular lipid metabolism. Our findings carry profound implications and can serve as a source of inspiration for the future design of biocompatible nanomaterials and nanomedicines.