Pyridostigmine bromide (PB), a highly hygroscopic drug was selected as the model drug. The PB sustained-release (SR) dosage forms that were developed by extrusion-spheronization, direct compression and fluid-bed methods. Produced by the aforementioned methods, the optimal PB-SR dosage forms could improve the hygroscopic defect of PB and reach the SR effect. In vivo study, a good linear regression relationship was observed between the fraction dissolution and fraction absorption for the optimal PB-SR dosage forms. However, the results of microdialysis technique shown that the release of acetylcholine and the release of protein unbound drug for the PB-SR pellets provided with SR effect in vivo as well.