Natural polysaccharides, as green biomacromolecules, possess unique potential in modulating immune function, mitigating oxidative damage and maintaining tissue homeostasis. The present study was conducted to explore the in vitro antioxidant activity and underlying mechanism of Siegesbeckia orientalis L. polysaccharide III (SIE-Ⅲ). The scavenging abilities of SIE-Ⅲ against DPPH radicals, ABTS cation radicals, hydroxyl radicals (·OH) and superoxide anion radicals (O2?·) were measured by chemical antioxidant assays. An oxidative stress model was constructed using H?O?-challenged RAW264.7 cells. The cytoprotective effects of SIE-Ⅲ were assessed in this cell model, and its impacts on oxidative stress-related factors, intracellular reactive oxygen species (ROS) levels, nuclear translocation of Nrf2, as well as the gene and protein expression associated with the Nrf2/Keap1/P62 signaling pathway were also investigated. It was shown that radical scavenging effects of SIE-Ⅲ in chemical systems were presented in a concentration-dependent manner. The IC50 values of SIE-Ⅲ for scavenging O2?·, ABTS?·, DPPH· and ·OH were 3.16, 2.18, 3.15 and 3.97 mg·mL?¹, respectively, which were higher than those of the positive control vitamin C (Vc). No cytotoxicity was detected for SIE-Ⅲ in the cellular experiment. The survival rate of H?O?-damaged cells was significantly elevated to 93.63% after SIE-Ⅲ intervention. Meanwhile, intracellular ROS levels were reduced obviously. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), together with glutathione (GSH) content were increased, whereas the generation of malondialdehyde (MDA) was suppressed (P<0.05). Mechanistic studies demonstrated that the mRNA and protein expression of P62, Nrf2, HO-1, NQO1, SOD1 and CAT were markedly upregulated by SIE-Ⅲ. The expression of Keap1 was downregulated, and the nuclear translocation of Nrf2 was facilitated simultaneously (P<0.05).In summary, the antioxidant effect of SIE-Ⅲ may be associated with the activation of the P62-Keap1-Nrf2 signaling pathway, and a theoretical basis is provided for the development of natural antioxidants.