Hydrophobic ion-pairing (HIP) complexation has emerged as an efficient approach to enhance the entrapment of therapeutic peptides in the biodegradable polymer matrix. In the present study, we developed an innovative extraction method for preparation of HIP-colistin (CST, a polycationic peptide) using various water-insoluble anionic lipids. To determine the loading mechanism of HIP-CST entrapped poly(lactic acid) (PLA) nanoparticles (HIP-CST-PLA-NPs), the effects of anionic lipids and PLA molecular weight (Mw) on the unentrapped fraction (uf) of CST in PLA-NPs were investigated. And CST release mechanism from HIP-CST-PLA-NPs was also investigated by evaluating their release behavior and NP swelling. It is showed that HIP-CST retention in the PLA-NPs was imposed by their physical localization in the networks of the PLA chains, rather than the electrostatic attraction between anionic lipid and CST in serum. And HIP-CST-PLA-NPs in serum exhibited the swelling-controlled release behavior with a substantially accelerated release and NP swelling observed in comparison with that in phosphate buffer. Our results can effectively guide the preparation of biodegradable polymer based modified drug release systems with desired properties for peptides delivery.