Dry eye is both a common symptom and a disease. It is known that the ophthalmic emulsion of the immunomodulator cyclosporine-A (Cyc-A) has a positive effect on this condition but its’ absorption to intraocular tissues is limited. Nanosuspension is a drug formulation that aims to increase the bioavailability. The aim of this study was to develop a Cyc-A nanosuspension formulation for a better intraocular absorption via ocular delivery and to investigate the effectiveness of the formulation by comparing it with two marketed ophthalmic emulsions (Restasis® and Depores®). Two types of Cyc-A loaded Eudragit S100 nanosuspension (A and B) were prepared. Drug formulations were applied to both eyes of 20 male Albino New Zealand rabbits with an interval of 12 hours for 14 days. In vitro drug release was tested using a dialysis sac and quantitative analysis was performed by HPLC for evaluating Cyc-A amounts in all formulations. Although all four formulations had similar particle size and polydispersity indexes, nanosuspension B which had a positive zeta potential value, had released more Cyc-A than other formulations. It was showed that Cyc-A loaded nanosuspension formulations which had good ocular tolerability could be a better alternative to commercial formulations for the treatment of dry eye. The nanosuspensions have ideal mean particle size range with a positive surface charge for ophthalmic applications of Cyc-A.