Abstract: OBJECTIVE To study the pharmacokinetics of dual conjugated chitosan-mitomycin C nanoparticles (CS-MMC-NPs) in rats. METHODS The two groups of rats were injected with CS-MMC-NPs and MMC injection at the dose of 4 mg·kg^-1. The concentrations of MMC in plasma at different time were determined by HPLC-MS and the main pharmacokinetic parameters were calculated. RESULTS The calibration curves were linear over the range of 20-1 000 μg·L^-1. The limit of quantitation was 20 μg·L^-1. The within day and day to day relative standard deviation(RSD) was <15%. The main pharmacokinetic parameters of CS-MMC-NPs and MMC injection were as follows: t_1/2 were (2.64±0.11)h and (0.49±0.049)h, AUC_0-∞ were (2.01±0.11)mg·h^-1·L^-1 and (0.93±0.075)mg·h^-1·L^-1, Vz were (1.52±0.18)L and (0.63±0.065)L, CL were (6.95±0.70)mL·min^-1 and (15.47±1.89)mL·min^-1. The differences of parameters were significant between two preparations. CONCLUSION The method is sensitive, accurate, specific for the pharmacokinetic study of CS-MMC-NPs. Compared with MMC injection, CS-MMC-NPs has a controlled releasing rate, a high level of blood concentrations and a long blood circulation time, which benefits the control of acute toxicity of MMC for the rats.