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This study was aimed to investigate the antioxidant effect of <i>Paeonia lactiflora</i> Pall. petal flavonoids extract (PPF) on <span style="font-variant: small-caps;">d</span>-galactose (<span style="font-variant: small-caps;">d</span>-gal)-induced ICR mice. In this study, sixty male ICR mice were randomly divided into six groups during an 8 weeks experimental period, including normal control (NC) group, <span style="font-variant: small-caps;">d</span>-gal group, epigallocatechin gallate (EGCG) group, low, medium, and high dose PPF groups (10, 20 and 40 mg/kg/day). The results showed that intragastric administration with PPF significantly reverses the atrophy of the visceral organs of oxidative damage mice in a dose-dependent relationship. PPF indicated the antioxidant capacity to decrease the malondialdehyde (MDA) level and improve the activity of superoxide dismutase (SOD), catalase (CAT) as well as glutathione peroxidase (GSH-Px). In addition, PPF treatment reversed gut microbiota dysbiosis by increasing the relative abundance of <i>Lactobacillaceae</i>. Spearman correlation analysis showed that the body’s oxidative stress markers were directly related to changes in gut microbiota. These findings reveal firstly that PPF could alleviate <span style="font-variant: small-caps;">d</span>-Gal-induced oxidative stress and modulate gut microbiota balance.