Polysaccharides are recognized as the predominant active constituents of Polygonatum cyrtonema Hua, conferring a broad spectrum of benefits. However, the structure-activity relationship of Polygonatum cyrtonema polysaccharides (PCP) and oligosaccharides (PCOP) remains underexplored. In this study, we aimed to compare the chemical properties and biological activities of PCP and PCOP. Our analysis revealed that both PCP and PCOP showed similar glycosidic linkages, characterized by linear →1)-β-Fruf (2 → units constituting the backbone with glucose. However, they differed significantly in molecular weight, with PCP measuring 1.78 × 10 Da and PCOP measuring 1.65 × 10 Da. Our findings showed that both PCP and PCOP could protect the intestinal barrier and regulate short-chain fatty acid levels. Notably, PCOP demonstrated superior efficacy in alleviating colitis symptoms and regulating inflammatory factors compared to PCP. Additionally, PCOP increased the relative abundance of Faecalibaculum apart from norank_f__Muribaculaceae in colitis mice more effectively than PCP. Collectively, these results suggest that the molecular weight of PCP and PCOP significantly influences their anti-inflammatory effects, thereby laying the groundwork for the future development and application of Polygonatum cyrtonema glycans in therapeutics or functional foods.