Protein nanoparticles represent a promising system for the encapsulation and delivery of bioactive compounds or highly sensitive ingredients. In this study, whey protein isolate nanoparticles (WPINs) were prepared by using supercritical carbon dioxide (scCO 2) treatment. The effects of scCO 2 on the size and stability of the WPINs were investigated. The average particle diameter ranged from 100 to 300 nm when the WPIN solution was treated with scCO 2 at 8–24 MPa and 45 °C for 30 min. ScCO 2 -treated WPINs had more uniform size distribution and smaller particles than WPINs prepared without scCO 2. However, particle aggregation was observed during heating to 55–75 °C. Fluorescence and differential thermogravimetric analysis showed that CO 2 pressure did not cause secondary and tertiary structure changes or denaturation of WPINs. The change in WPINs size was likely caused by the high diffusibility of scCO 2 , which could slightly modify WPINs local structure, affecting hydrophobic interactions and hydrogen bonding within the molecule. These results indicate that the size of nano-particles can be modulated by adjusting CO 2 pressure, which may facilitate WPINs use in food and biomedical products. • Whey protein isolate nanoparticles (WPIN) were prepared using supercritical carbon dioxide (scCO 2) treatment. • The impact of scCO 2 treatment on the structures of WPINs. • The size of the nanoparticles can be regulated by adjusting the CO 2 pressure. [ABSTRACT FROM AUTHOR]