Faster biodegradation, immunogenicity and lack of cell penetrative capabilities are hurdles in development of peptidyl drugs for cancer therapy. Polymeric carriers can be used to overcome these problems. The present study is focused on the use of polyhydroxybutyrate as a potential nanovehicle for the delivery of anticancer peptides. PHB (72 kDa) was produced by thermal treatment of high molecular weight PHB (300 kDa) under melt conditions and then conjugated with PEG (4 kDa) by Steglich esterification reaction. Anticancer peptide NuBCP-9 (FSRSLHSLL) encapsulated PHB72K-PEG4K NPs were prepared by double emulsion solvent evaporation method. PHB72K-PEG4K NPs showed encapsulation efficiency of 61% and exhibited sustained release of peptide over a period of 26 days at physiological pH. NuBCP-9 loaded PHB72K-PEG4K NPs showed an IC50 value of 2.2 μM & 1.6 μM in MCF-7 cells in 48 h and 72 h respectively. Confocal laser microscopy confirmed efficient cellular uptake and induction of apoptosis by peptide loaded NPs in a time dependent manner. In vivo intraperitonial administration of 20 mg/kg NuBCP-9/NPs twice a week for three weeks triggered 90% tumor regression in Ehrlich syngeneic mouse model. Our results illustrated the potential of PHB72K-PEG4K based nanoformulation as a tool for targeting intracellular proteins.