A sulfated polysaccharide consisting of two components with molecular weights of 439 kDa and 16 kDa was extracted from the salt brine. The structural properties, immunomodulatory activity, in vitro fermentation behaviors, and effects of SP on regulating the gut microbiota were investigated. The chemical composition and monosaccharide composition analysis showed that the neutral sugar, protein, uronic acid, and sulfated group contents of SP were 60.42 ± 0.04%, 2.90 ± 0.01%, 13.34 ± 0.01% and 10.51 ± 0.01%, respectively, containing arabinose, galactose, glucose, rhamnose, xylose, mannose, and glucuronic acid in a molar ratio of 33.24:19.18:16.64:13.25:8.31:4.11:5.27. Results from the macrophage cell model showed that SP intervention improved the proliferation activity, phagocytosis of neutral red, and production of IL-6 and TNF-α in RAW 264.7. Furthermore, in vitro fermentation of SP by gut microbiota showed that SCFA production in all treatment groups was significantly higher than that of the blank control group after 48 h of fermentation, especially butyric acid which was 1.70 folds that of the control group. Moreover, long-term fermentation (48 h) of SP improved the diversity of microbiota, decreased the F/B ratio (30.75 at 0 h vs. 1.22 at 48 h), and promoted the growth of probiotics (Parabacteroides, Bacteroidetes, Ruminococcaceae, and Phascolarctobacterium). The positive regulatory effect of SP on the gut microbiota and its metabolites is considered a potential target for its immunomodulatory activity.Graphical abstract