Oxidative stress is considered as a critical factor in the process of pathological diseases, and mitochondria are considered as vital target organelles for disease intervention. The purpose of this study was aimed to evaluate the antioxidant efficacy of mitochondria-targeted astaxanthin nanoparticle on hydrogen peroxide-induced oxidative damage. As expected, mitochondria-targeted nanoparticle showed excellent mitochondria co-localization ability with higher Pearson’s correlation coefficient (r = 0.88). In vitro experiments suggested that the mitochondria-targeted astaxanthin nanoparticle could promote cell viability and increase antioxidant-related enzyme activities. Simultaneously, metabolomics analysis indicated that mitochondria-targeted astaxanthin nanoparticle could alleviate oxidative stress by regulating amino acid metabolism and energy metabolism. Altogether, all these results strongly confirmed the mitochondria-targeted strategy for astaxanthin delivery could relieve oxidative stress and had great promise in the application of disease intervention.