Rapamycin-loaded chitosan/polylactic acid nanoparticles with size of about 300 nm in diameter were prepared through nanoprecipitation method using cholesterol-modified chitosan as a stabilizer. The surface coating of chitosan, which was demonstrated by zeta potential measurement, endowed the nanoparticles good retention ability at the procorneal area, facilitating the sustained release of rapamycin on the corneal. The immunosuppression in corneal transplantation of the nanoparticles was investigated using rabbit as animal model, the median survival time of the corneal allografts treated with nanoparticles was 27.2+/-1.03 days and 50% grafts still remained surviving by the end of the observation, while the group treated with 0.5% rapamycin suspension was 23.7+/-3.20 days. The median survival time of drug-free nanoparticles group and untreated groups were 10.9+/-1.45 and 10.6+/-1.26 days, respectively. The results demonstrated the excellent immunosuppression of rapamycin-loaded chitosan/polylactic acid nanoparticles in corneal transplantation.