The purpose of this research was the fabrication, statistical optimization, and in vitro characterization of insulin-loaded poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanoparticles (INS-PHBV-NPs). Nanopar-ticles were successfully developed by double emulsification solvent evaporation method. The NPs were characterized for particle size, entrapment efficiency (EE%), and polydispersity index (PDI). The NPs also were characterized by scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and circular dichroism (CD). The optimum conditions were found to be 1.6% polyvinyl alcohol (PVA), 0.9% of PHBV, and 15 mg/ml of insulin with the aid of the Box-Behnken experimental design results. The optimized NPs showed spherical shape with particle size of 250.21 ± 11.37 nm, PDI of 0.12 ± 0.01, and with EE% of 90.12 ± 2.10%. In vitro drug release pattern followed Korsmeyer-Peppas model and exhibited an initial burst release of 19% with extended drug release of 63.2% from optimized NPs within 27 d. In conclusion, these results suggest that INS-PHBV-NPs could be a promising candidate for designing an injectable sustained release formulation for insulin.