Updated project metadata. Upon entering in contact with the biological environment, nanostructures are immediately covered by biomolecules, and in particular proteins forming the so-called “protein corona” (PC). The phenomenon of PC formation has gained great attention in recent years due to its implication for the use of nanostructures in biomedicine. In fact, it has been shown that the formation of the PC can impact the performance of nanostructures by reducing their stability, causing aggregation, increasing their toxicity, and causing unexpected and undesired nanostructure-cell interactions. In this work, we decided to study for the first time the formation and the evolution of PC on the surface of nanostructured lipid carriers loaded with superparamagnetic iron oxide nanoparticles (SPIONs), before and after the crossing of an in vitro model of the blood-brain barrier. Combining confocal microscopy, direct STochastic Optical Reconstruction Microscopy (dSTORM), and proteomic analysis, we were able to provide a complete analysis of the PC formation and evolution. In particular, we evidence that PC formation is a fast process, being formed around particles even after just 1 min of exposure to fetal bovine serum. Moreover, PC formed around particles is extremely heterogeneous: while some particles have no associated PC at all, others are completely covered by proteins. Lastly, the interaction with an in vitro blood-brain barrier model strongly affects the PC composition: in particular, a large amount of the proteins forming the initial PC is lost after the BBB passage and is partially replaced by new proteins derived from both the brain endothelial cells and from the cell culture medium. Altogether, the presented data could potentially provide new insights into the design and fabrication of lipid nanostructures for the treatment of central nervous system disorders.