Arthrospira platensis polysaccharide component 1 (PAP-1), a purified polysaccharide monomer isolated from Arthrospira platensis, exhibits pronounced antioxidant activity. To investigate the in vivo and in vitro regulatory effects of PAP-1 on antioxidant enzyme activities and inflammatory mediators in mice and RAW264.7 cells, the mice were administered PAP-1 by gavage, and the cells were cultured with PAP-1. Subsequently, serum, lung, spleen, and thymus tissues from mice, as well as the cultured RAW264.7 cells, were collected for analysis using RNA sequencing, commercial assay kits, immunohistochemistry, RT-qPCR, and Western blotting. The results demonstrated that PAP-1 significantly reduced the levels of oxidative stress-related indicators (NO, iNOS, MDA, MPO, and XOD), while markedly enhancing the activities of antioxidant enzymes (SOD, CAT, and GSH-Px) (p < 0.05), a trend consistently observed in both in vivo and in vitro experiments. Furthermore, PAP-1 upregulated the expression of key antioxidant genes and proteins, including HO-1, NQO1, GCLM, p62, Prdx1, and SLC7A11. Collectively, these findings indicate that PAP-1 exerts regulatory antioxidant effects in mice and RAW264.7 cells by enhancing antioxidant enzyme activity and suppressing oxidative stress responses, underscoring its potential as a natural antioxidant agent.