Interferon gamma (IFN-γ) challenges the ability of viruses to distinguish between target and off-target cells i.e. viral tropism. Due to the similarity of viruses and nanoparticles in shape and size, we hypothesized that the effect of IFN-γ on viruses also applies to virus-mimetic nanoparticles. Therefore, we determined the influence of IFN-γ on nanoparticle tropism and cell uptake. For our experiments, we used cyclic RGD-peptide (cRGD) functionalized polymeric nanoparticles and lipid nanocapsules (LNC) to target the αvβ3 integrin receptor on cell membranes of rat mesangial cells (rMCs), HeLa cells and human dermal microvascular endothelial cells (HDMEC). We used a nanoparticle concentration range between 0.1 and 2 nM. The IFN-γ concentrations and IFN-γ incubation times varied in the range of 100 IU–1000 IU and zero to 54 h respectively. When we measured nanoparticle uptake via flow cytometry 24 h after cytokine administration to cells, our findings confirmed that IFN-γ decreased the tropism as well as the uptake of functionalized nanoparticles by target cells. We were able to demonstrate a maximal IFN- γ effect at a concentration of 100 IU of IFN-γ and a 24-h incubation time. The presence of IFN-γ is associated with an upregulation of cholesterol 25-hydroxylase (CH25H). That the CH25H oxidation product, 25-hydroxycholesterol (25HC), is known to inhibit cell-nanoparticle interactions points at the potentially underlying mechanism of the observed INF-γ effect.