Very small superparamagnetic iron oxide nanoparticles (VSOPs) show diagnostic value in multiple diseases as a promising MRI contrast agent. Macrophages predominantly ingest VSOPs, but the mechanism remains unclear. This study identifies differences in VSOP uptake between pro-inflammatory M1 and anti-inflammatory M2 macrophages and explores the role of the pericellular glycocalyx. Glycosaminoglycans (GAG) synthesis activities and the pericellular glycocalyx for M1/M2-like macrophages were assessed by RT-qPCR, Click-iT reaction, and WGA-FITC staining. The uptake of europium-VSOP and Synomag by the two subtypes was measured using Prussian blue staining, fluorescent microscopy, and magnetic particle spectroscopy. The findings revealed that M2-like macrophages had higher GAG synthesis activity, a thicker glycocalyx, and increased nanoparticle uptake compared to M1-like macrophages. Enzymatic glycocalyx degradation significantly decreased nanoparticle uptake. This study demonstrates a positive correlation between glycocalyx and nanoparticle uptake that could be exploited for imaging and targeted therapy, particularly in cancer, where macrophage subtypes play distinct roles.