Saponification contributed to an increase in the in vitro antioxidant activity of astaxanthin (Asta) extracts derived from Penaeus sinensis (Solenocera crassicornis) by-products. However, the influence of non-saponification (N-Asta) and saponification Asta (S-Asta) absorption on antioxidant activity in vivo was limited. The antioxidant properties of N-Asta and S-Asta were therefore compared in Sprague Dawley male rats after 6 h and 12 of absorption using biochemistry assays combined with an untargeted metabonomics strategy. Non-saponified Asta and S-Asta showed similar digestive properties in a stimulated gastrointestinal tract. Increased glutathione content and decreased malondialdehyde content were measured in the liver tissues of N-Asta and S-Asta treated rats after 12 h of absorption. Absorption of N-Asta increased liver total superoxide dismutase, glutathione peroxidase, and catalase activity. Treatment with S-Asta up-regulated NAD(P)H: quinine oxidoreductase-1, and heme oxygenase-1 expression was associated with the nuclear erythroid 2-related factor 2/antioxidant responsive element pathway at the end of 12 h absorption. With partial least square-discriminant analysis and metabolite heatmap profiles, the S-Asta group was clearly separated from the N-Asta group. The S-Asta treatment also demonstrated stronger influences on plasma metabolites than the N-Asta treatment. Both N-Asta and S-Asta absorption showed critical roles in the regulation of specific metabolites, and 15 potential biomarkers were identified in eight key pathways to separate these experimental groups after 12 h of absorption. However, an increased serotonin level was only detected in the S-Asta group after 12 h absorption. Absorption of N-Asta and S-Asta induced different antioxidant effects in normal rats, which were associated with metabolite changes. © 2022 Society of Chemical Industry.