The aim of our study was to prepare and characterize biodegradable insulin-loaded solid lipid nanoparticles (Ins-SLNs) flocculates for the pulmonary delivery of Ins. The cationic Ins-SLNs and anionic Ins-SLNs were prepared by W/O/W emulsion technique, respectively. Then anionic Ins-SLNs were self-assembled into flocculates via electrostatic interactions with the oppositely surface charged cationic Ins-SLNs followed by lyophilized into dry powders. The maximal entrapment efficiency of cationic Ins-SLNs and anionic Ins-SLNs were 56.32 +/- 1.01% and 66.02 +/- 1.58%, respectively. Freeze-drying the Ins-SLNs flocculates yielded dry powders with desirable aerodynamic diameter of 2.04 +/- 0.17 microm and low bulk density of 0.06146 +/- 0.0045 g/cm3, suitable for inhalation. In addition, the flocculates showed high aerosolization efficiency (emitted fraction of 92.54 +/- 0.77% and respirable fraction of 66.89 +/- 3.02%). The in vivo study showed that Ins-SLNs flocculates could prolong hypoglycemic effect and a relative pharmacological bioavailability of 35.62 +/- 1.34% could be achieved after intratracheal instillation to diabetic rats at the dose of 8 IU/kg dosage. Therefore, the biodegradable SLN flocculates fabricated via charge interaction may provide a useful strategy to fabricate dry powder for pulmonary administration of protein therapeutics or antigens.