Objective：To study the pharmacokinetics of pyridostigmine bromide and oil-in-water pyridostigmine bromide novel phos－pholipids complex nanoemulsion（PPCN） in rats，and to compare their relative bioavailability and bioequivalence. Methods：In this study，twelve rats were randomly divided into two groups and the same dose was administered. Blood samples was taken at different time points，and the plasma concentrations of the drugs were determined by HPLC. The DAS（2.1.1） software were used to calculate the main pharmacokinetic parameters and bioequivalence. Results：The Cmax of PB and PPCN were 1.49 mg/L and 2.23 mg/L，re－spectively. The AUC（0-72 h） of PB and PPCN were 7.06 mg·h/L and 14.12 mg·h/L respectively. The Tmax of PB was 1.5 h，and the Tmax of PPCN was 4 h. The Cmax of PPCN was 1.50 times that of PPCN，and the AUC（0-72 h） of PB was 2 times that of PPCN. The results of AUC（0-72 h） Cmax and Tmax were statistically different（P values were 0.000，0.000，0.001，respectively） by student’s t test，analysis of variance and wilconxon rank sum test. Conclusion：PPCN can not only prolong the action time of PB in rats，but also improve oral bioavailability of PB in rats，and PPCN and PB are not bioequivalent，and PPCN is better than PB.