The aim of this work was to study the influence of the concentration and molecular weight ofpoly (DL-lactide)(PLA)on the characteristics and in vivo biological activ-iry of protein-loaded microspheres. At the same time, an attempt was made to achieve further optimization of the formulation. In the stuay, insulin was choosen as a model of protein drugs. Nine formulations of injectable insulin-loaded PLA microspheres were prepared using all emulsification and solvent evaporation pro-cess according to a factorial design. The trapping efficiency, drug loading, and the drop percentages of blood glucose levels at 24 hr and 72 hr in mice were used to evaluate the foFmulations. The results showed that PLA molecular weig•ht and, especially, PLA concentration exerted influences on the characteristics and in vivo biological activity of insulin. 10aded microspheres. The drug-trapping efficiency in-creased with the increase ofthe polymer concentration. The drug loading decreased with the increase of the polymer concentration and was not obviously affected by PLA molecular weight. The drop percentage of blood glucose level at 24 hr in-creased with the increase of polymer concentration and molecular weight. At 72 hr, the drop percentages of blood glucose levels were slightly increased with the increase of PLA concentration and then significantly decreased after the PLA con-centration was above 150 mg/ml An optimizedformulation was prepared with PLA-