The physicochemical properties, structural characteristics, antioxidant, radioprotective and lipid-lowering activities, as well as the underlying structure-activity relationships of polysaccharides extracted from Nostoc flagelliforme grown under normal (WL-EPS-1), salt stress (NaCl-EPS-1) and mixotrophic culture conditions (Glu-EPS-1) were studied. The results demonstrated that WL-EPS-1, NaCl-EPS-1 and Glu-EPS-1 were heteropolysaccharides comprising different proportions of monosaccharides and uronic acid, with different average molecular weights of 0.99 × 103, 1.09 × 103 and 1.18 × 103 kDa, respectively. Their intrinsic viscosity were significantly different, at 24.72, 29.98, and 41.06 dL/g, respectively. The functional groups of polysaccharides were not greatly affected, but the chemical composition, triple-helix structure, chain length and surface morphology were significantly influenced by culture conditions. In vitro bioactivity assays showed that the antioxidant activity generally increased in the order of WL-EPS-1 < NaCl-EPS-1 < Glu-EPS-1, while NaCl-EPS-1 had the best radioprotective effect, and Glu-EPS-1 had the best lipid-lowering effect. In addition, the structure-activity relationship of the polysaccharides was analyzed by partial least squares, which revealed that the most important factors affecting the antioxidant, radioprotective and lipid-lowering activities of polysaccharides were viscosity and molecular weight. This study provides a strategy for obtaining high-bioactivity polysaccharides by appropriate regulation of culture conditions, as well as opening new directions for the molecular modification of polysaccharides.