In this study, polysaccharides from Sargassum fusiforme (SFP) were obtained by cellulase assisted hot water extraction. The chemical composition, structural characteristics, and in vitro fermentation properties of SFP were investigated. Results showed that the contents of total carbohydrate, protein, uronic acid and sulfate in SFP were 83.25%, 1.42%, 12.80% and 7.81%, respectively. It mainly consisted of fucose glucose and galactose, with molecular weight of 255.83 kDa. UV spectrum, FTIR, SEM and AFM results showed that SFP was a typical sulfate polysaccharide with relative smooth surface and regular shape. After in vitro fermentation for 24 h, the pH value of fermentation medium declined significantly (p < 0.05), utilization of carbohydrate was 53.17%. The contents of total SCFAs increased by 10.77 times. Moreover, SFP fermentation could change obviously the microbiota composition. It significantly increased the abundance of Faecalibacterium (increased by 49.07% compared with the Blank24 group), Phascolarctobacterium (increased by 88.06%), Bifidobacterium (increased by 139.13%), Ruminococcaceae_UCG-014 (increased by 177.78%), and Lactobacillus (increased by 400.00%), decreased the abundance of Prevotella_9 (decreased by 34.54%) and Blautia (decreased by 40.79%) at genus level. These results showed that SFP could be utilized by microbiota in human feces, and may have the potential to improve intestinal health. Display omitted •SFP was mainly composed of fucose, glucose and galactose, with MW of 255.83 kDa.•SFP (53.17%) was utilized by microbiota to produce 115.75 mM SCFAs at 24 h.•SFP intervention could change the diversity of gut microbiota.•Bifidobacterium and Lactobacillus were increased significantly in SFP group.