The limited bioavailability of β-carotene hinders its potential application in functional foods, despite its excellent antioxidant properties. Protein-based nanoparticles have been widely used for the delivery of β-carotene to overcome this limitation. However, these nanoparticles are susceptible to environmental stress. In this study, we utilized glycosylated oat protein isolate to prepare nanoparticles loaded with β-carotene through the emulsification-evaporation method, aiming to address this challenge. The results showed that β-carotene was embedded into the spherical nanoparticles, exhibiting relatively high encapsulation efficiency (86.21 %) and loading capacity (5.43 %). The stability of the nanoparticles loaded with β-carotene was enhanced in acidic environments and under high ionic strength. The nanoparticles offered protection to β-carotene against gastric digestion and facilitated its controlled release (95.76 % within 6 h) in the small intestine, thereby leading to an improved in vitro bioavailability (65.06 %) of β-carotene. This improvement conferred the benefits on β-carotene nanoparticles to alleviate tert-butyl hydroperoxide-induced oxidative stress through the upregulation of heme oxygenase-1 and NAD(P)H quinone dehydrogenase 1 expression, as well as the promotion of nuclear translocation of nuclear factor-erythroid 2-related factor 2. Our study suggests the potential for the industry application of nanoparticles based on glycosylated proteins to effectively deliver hydrophobic nutrients and enhance their application.