The insulin-loaded poly-L-lactide porous microspheres (INS-PLLA PMs) were successfully developed in an emulsion-combined precipitation of compressed CO2 antisolvent (PCA) using ammonium bicarbonate (AB) as a porogen. The resulting INS-PLLA PMs exhibited a rough and porous structure with a geometric mean diameter (D-g) of 15.62 mu m, an aerodynamic diameter (D-a) of 4.31 mu m, a fine particle fraction (FPF) of 65.57% and good aerosolization characteristics. The physicochemical characterization reveals that no chemical changes occurred on INS-PLLA PMs, while minor structural changes existed in insulin. The result of circular dichroism (CD) spectroscopy demonstrates a slight change happened in the secondary structure of insulin, however, the bioactivity verification test shows that the hypoglycemic activity of insulin from INS-PLLA PMs was well maintained, which shows no significant difference from the raw insulin. The fluorescent image of INS-PLEA PMs demonstrates that the insulin was homogeneously distributed in the matrix, and INS-PLEA PMs displayed a sustained-release effect. Furthermore, INS-PLLA PMs with almost no organic residue could promote the safety and suitability for pulmonary delivery of protein drugs. This study indicates that emulsion-combined PCA process is an effective and benign technology to produce INS-PLLA PMs, which have potential in the application of pulmonary drug delivery for treatment of diabetes. (C) 2015 Elsevier B.V. All rights reserved.