Cyclosporine A is a calcineurin inhibitor and is usually used as an immunosuppressant medication. The main purpose of this study is to develop nanosuspension of polypeptide cyclosporine A by using the wet milling method for oral administration. Cell culture studies were also performed with human intestinal Caco-2 cell lines. Hydroxypropyl methylcellulose and sodium dodecyl sulfate were used as stabilizers in nanosuspension. In vitro characterization studies such as Fourier-transform infrared analysis and morphological imaging with scanning electron microscopy have been carried out with obtained cyclosporine A nanosuspension. The particle size, particle size distribution, and zeta potential values of the nanosuspension were measured approximately 400 nm, 0.4, and -25 mV, respectively. The solubility of cyclosporine A was increased 4.5 times in nanosuspension compared to the coarse cyclosporine A powder. As a result of cytotoxicity studies conducted with different concentrations, it was decided to conduct permeability studies at a dose equivalent to 150 mu g/mL cyclosporine A. Permeation studies have shown that the nanosuspension increases cyclosporine A transport by 5 and 1.5 times, respectively, compared to coarse powder and commercial product.