Background: Catharanthus roseus (CR) shows promising anticancer activity. However, there is limited information on its polymeric formulation. Objectives: Therefore, current study aimed to characterize poly(lactic-co-glycolic acid (PLGA) CR nanoparticles and determine its effects on resistant human epidermal receptor 2 (HER2)-overexpressed breast cancer cells. Methods: PLGA-polyethylene glycol (PEG) CR nanoparticles were synthesized using the solvent displacement method and characterized using ultraviolet-visible spectroscopy (UV-VIS), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering, transmission electron microscopy (TEM), zeta potential, encapsulation efficiency, and drug release experiments. Cytotoxicity was done using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Protein expression was done with a gel image analyzer. Cell morphological changes were viewed under phase-contrast microscope. Results: TEM images showed the nanoparticles were spherical and size less than 100 nm. FTIR results indicated encapsulation of CR based on the presence of 3327 cm–1, 1637 cm–1, and 1066 cm–1 peaks. Encapsulation efficiency was >60% in both formulations. However, pluronic F68 PLGA-PEG CR nanoparticles showed a gradual release of CR compared with polyvinyl acetate (PVAc). The cytotoxicity assay showed that the half-maximal inhibitory concentration of the CR nanoparticles generated with F68 and PVAc was lower (42–58 μg/mL) on tamoxifen-resistant cells compared with parent cells (99–147 μg/mL). Further analysis using CR nanoparticles with F68 exhibited downregulation of HER2 expression and induced apoptotic features based on morphological changes. Conclusion: These findings suggest that PLGA-PEG nanoparticles could retain the cytotoxic effects of CR.