Cyclosporin A nanocrystals (CsA-NCs) interaction with Caco-2 cells were investigated in this study, including cellular uptake and transport across Caco-2 cell monolayers. CsA-NCs of 165 nm, 240 nm and 450 nm were formulated. The dissolution of CsA-NCs was investigated by paddle method. The effect of size, concentration and incubation time on cellular uptake and dissolution kinetics of CsA-NCs in cells were studied. Uptake mechanisms were also evaluated using endocytotic inhibitors and low temperature (4 °C). The cell monolayers were incubated with each diameter CsA-NCs to evaluate the effect of size on the permeation characteristics of CsA across the intestinal mucosa. The results of dissolution study showed that 165 nm CsA-NC had the highest dissolution rate followed by 240 CsA-NC and finally 450 nm CsA-NC. The saturation of cell uptake of CsA-NCs was observed with the increase of incubation concentration and time. 240 nm and 450 nm CsA-NCs had the lowest and highest uptake efficiency at different time and drug concentration, respectively. The uptake of all three-sized CsA-NCs declined significantly in some different degree after the pre-treatment with different endocytosis inhibitors. 165 nm CsA-NC showed a highest transport capacity across monolayers at the same concentration and time. The results suggest that the size of CsA-NCs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. Decreasing particle size of CsA-NCs can improve transport capacity of CsA through cell monolayer.