Nanostructured lipid carriers (NLCs) are generally recognized as safe (GRAS) to form a controlled nanostructure are a new generation of lipid nanoparticles. In addition to formulation and particle surface properties, particle size had great influence for overcoming gastrointestinal (GI) barriers on the oral drug delivery of lipid based nanoparticles. In the present study, we investigated the effect of size on oral drug delivery for NLCs. The NLCs with different particle sizes (NLCs 100 nm, NLCs 200 nm and NLCs 300 nm) were prepared by using solvent evaporation method and the coumarin-6 (C6) or DiO/DiI was loaded in the nanoparticles as the fluorescence probe. The MTT assay indicated that both blank NLCs and C6-loaded NLCs displayed relatively low toxicity towards Caco-2 cells. Cellular uptake mechanisms of NLCs with different sizes were found to be similar and governed by active endocytosis, clathrin- and caveolae-mediated process. However, the smaller nanoparticle (NLC–100 nm) showed higher uptake efficiency in Caco-2 cell (P < 0.05) as well as higher permeation ability in Caco-2 cell monolayer (P < 0.01), compared with NLC–200 nm and NLC–300 nm. The fluorescence resonance energy transfer (FRET) assay indicated that all of three NLCs could maintained the structural integrity in blood circulation after oral administration. NLC–100 nm exhibited the most stability according to the most stable FRET signal. In situ rat intestinal absorption experiments and in vitro ligated rat intestinal loops model demonstrated that all NLCs could rapidly penetrate duodenum versus jejunum, ileum and colon (P < 0.01). Moreover, pharmacokinetic studies showed that NLC–100 nm exhibited highest Cmax and AUC compared with the other two NLCs. In conclusion, the size of 100 nm might be the most suitable size for oral drug delivery of lipid based nanoparticles.