Liquids containing nanoparticles (nanofluids) exhibit different spreading or thinning behaviors on solids than liquids without nanoparticles. Previous experiments and theoretical investigations have demonstrated that the spreading of nanofluids on solid surfaces is enhanced compared to the spreading of base fluids without nanoparticles. However, the mechanisms for the observed enhancement in the spreading of nanofluids on solid substrates are not well understood. The complex nature of the interactions between the particles in the nanofluid and with the solid substrate alters the spreading dynamics [Wasan, D. T.; Nikolov, A. D. Nature 2003, 423, 156]. Here, we report, for the first time, the results of an experimental observation of nanoparticles self-structuring in a nanofluid film formed between an oil drop and a solid surface. Using a silica-nanoparticle aqueous suspension (with a nominal diameter of 19 nm and 10 vol %) and reflected light interferometry, we show the nanoparticle layering (i.e., stratification) phenomenon during film thinning on a smooth hydrophilic glass surface. Our experiments revealed that the film thickness stability on a solid substrate depends on the film size (i.e., the drop size). A film formed from a small drop (with a high capillary pressure) is thicker and contains more particle layers than a film formed from a large drop (with a lower capillary pressure). The data for the film-meniscus contact angle verses film thickness (corresponding to the different number of particle layers) were obtained and used to calculate the film structural energy isotherm. These results may provide a better understanding of the complex phenomena involved in the enhanced spreading of nanofluids on solid surfaces.