Drug delivery systems based on star-shaped poly(lactic-co-glycolic acid) (PLGA) exhibit unique features such as better stability, smaller size distribution and higher drug capacity in comparison with linear PLGA. In this study, six-arm star-shaped PLGA was synthesized by ring-opening polymerization, and the structure was characterized by carbon-13 nuclear magnetic resonance spectroscopy and infrared spectroscopy. Puerarin (PU), a promising anti-Parkinsonism natural product, was encapsulated into six-arm star-shaped PLGA nanoparticles (PU-NPs) and thus to be potentially efficacious for the treatment of Parkinson’s disease (PD). The obtained PU-NPs were monodispersed with an average diameter of 88.36 ± 1.67 nm and a PDI of 0.047 ± 0.007 in water, which was further confirmed by transmission electron microscopy (TEM). In terms of the in vitro release, PU was released more slowly and more steadily from PU-NPs than from linear PLGA nanoparticles. Molecular dynamics and the coarse-grained dissipative particle dynamic method revealed the mechanistic details of six-arm star-shaped PLGA nanoparticles formation and PU loading and release, wherein the hydrophobic interaction was the driving force of nanoparticles formation and drug loading. The hydrophobic-hydrophilic transition of PU particles in changing of external environment was the fundamental driving force behind drug release. The PU-NPs could be internalized intact into Madin-Darby Canine Kidney (MDCK) cells, and the cellular uptake and permeability of PU-NPs were much higher than those of the raw PU (p<0.01). In addition, the PU-NPs afforded a significant neuroprotective effect against 1-methyl-4-phenylpyridinium ion (MPP+)-induced damage in SH-SY5Y cells. Meanwhile, the zebrafish embryos and larvae treated with PU-NPs did not show any obvious toxic effects, as evidenced by the unchanged morphology, mortality, hatching, and heart rate. Fluorescence resonance energy transfer (FRET) imaging in zebrafish suggested that intact nanoparticles could be quickly absorbed into the blood circulation and then reached the brain. When the PU-NPs were orally administrated to rats, the plasma Cmax and AUC0-t were found to be 1.96 ± 0.21 μg/ml and 27.47 ± 2.78 μg·h/ml, respectively, which were 6.1- and 10.5-fold higher than those measured in the unformulated PU. Brain delivery of PU was also much more effective with PU-NPs than with the unformulated PU. Most importantly, the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD mice had notably improved behavior disorder, reversed dopamine depletion, and attenuated the decrease in the levels of dopamine and its metabolites during one-week post-observation after treatment with PU-NPs. The protective effect was partially mediated by the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. The results suggested that star-shaped PLGA nanoparticles may be promising for application of brain delivery in PU to treat PD.