This study focused on exploring the potential value of Cucumis sativus L. seeds by isolating, purifying their polysaccharides, clarifying their basic structural characteristics, and evaluating their in vitro anti-inflammatory activity. To achieve this goal, Cucumis sativus seed polysaccharides (CSP) was first extracted using hot water extraction method and then precipitated with ethanol. For further purification, CSP was sequentially processed through a tandem column system consisting of Amberlite FPA90Cl anion-exchange resin and Amberlite FPC3500H cation-exchange resin, followed by additional purification on an ion-exchange cellulose DEAE Cellulose-52 column, which successfully yielded a homogeneous polysaccharide fraction designated as CSP-S8 with a yield of 7.05%. The structural characteristics of CSP-S8 were systematically analyzed using multiple techniques, such as chromatography, mass spectrometry, to identify its functional groups and glycosidic linkages. Additionally, in vitro cell experiments were performed on RAW 264.7 macrophages to assess the effects of CSP-S8 on cell viability and its anti-inflammatory properties, as well as the expression of inflammation-related genes. The results indicated that CSP-S8 had an average molecular weight of approximately 90 673 Da and was composed of rhamnose, galacturonic acid, glucose, galactose, and arabinose with a molar ratio of 7.6∶11.6∶14.1∶15.1∶16.1. FT-IR spectroscopy confirmed that CSP-S8 is an acidic polysaccharide containing α-glycosidic linkages. Cell experimental data showed that CSP-S8 significantly improved the viability of RAW 264.7 cells compared with the control group; moreover, it effectively inhibited lipopolysaccharides (LPS)-induced nitric oxide (NO) production and reduced the secretion of inflammatory cytokines including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in a dose-dependent manner when compared with the LPS-induced model group. RT-qPCR analysis further revealed that CSP-S8 remarkably downregulated the LPS-induced mRNA expression levels of TNF-α, IL-1β, IL-6, IL-10, and iNOS. In this study, the CSP‑S8 was successfully isolated and purified from CSP, and its structure was preliminarily characterized. It was confirmed that this polysaccharide could significantly improve the viability of RAW 264.7 cells in vitro, inhibit LPS‑induced NO production and the release of inflammatory factors, and down‑regulate the expression of related inflammatory genes, showing favorable anti‑inflammatory activity in vitro.