Background: In the treatment of lung diseases, drug showed low bioavailability and efficacy by conventional administration methods. Passive lung-targeting microspheres provide a method to deliver drugs from the vascular side, there is still a paucity of systematic studies on the biocompatibility of biodegradable-polymer microspheres. Results: We used poly (lactic acid-glycolic acid) (PLGA) microspheres with different particle sizes (3, 10, 25, and 40 mm) as a model. The optimal lung-targeting particle size of the PLGA microspheres is 10 mm and the corresponding range of maximum tolerated dose is 125-150 mg/kg. We hypothesized that the decrease of blood oxygen saturation under treatment of microspheres was caused by pulmonary embolism. We found varying degrees of blood circulation loss of lungs by micro computed tomography test, which indicated severe pulmonary embolism subsequent to intravenous injection of microspheres with a particle size >25 mm. Furthermore, we found lung injury and microspheres leaked from the blood vessels into the alveoli by H&E staining. This process was likely induced by increased secretion of matrix metalloproteinases (MMPs), which destroyed the alveolar-capillary barrier and trigger an inflammatory cascade. Finally, we found that optimal particle size and dose conditions did not affect normal physiological activities after the microspheres degraded. The upregulation of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) induced vessel recanalization and reestablishment, which promoted the progress of lung repair. Conclusions: Collectively, our results highlight the importance of accurately designing the optimal size and dose of microspheres for passive lung-targeting delivery.