To develop a novel ciclosporin A (CsA)-loaded nanosuspension causing less ocular irritation, a range of nanosuspensions was prepared with various polymers using a media milling method. The effects of polymer, milling time, milling speed and bead material on the particle size of the nanosuspension were investigated. Stability and irritation tests in rabbits' eyes were then performed comparing the nanosuspension with a commercial product. Of the nanosuspensions prepared with various polymers, that with PVA showed no creaming or sedimentation phenomena and gave the smallest particles of about 530 nm. The particle size decreased abruptly as the milling time increased to 2 h and then hardly decreased further. As the milling speed was increased, the particle size of CsA in the nanosuspension also increased. Nanosuspensions prepared with zirconia beads gave significantly finer particles than those with polystyrene beads. In particular, the CsA-loaded nanosuspension with a CsA/PVA/water weight ratio of 0.5/1/100 prepared using the top-down media milling method with zirconia beads of 300 microm diameter at 1000 rpm for 2 h gave a minimum particle size of about 530 nm. This nansuspension was physcally and chemically stable for at least two months. In the Draize test, both this nanosuspension and the commercial product gave very slight ocular irritation. However, in the Schirmer tear test, this nanosuspension caused less irritation to the rabbits' eyes compared to the commercial product. Thus, the CsA-loaded nanosuspension prepared with PVA and water using the top-down media milling method could be a promising candidate for causing less ocular irritation.