The aim of the present study was to investigate structural characteristic and in vitro fermentation of a novel polysaccharide named ST-P2 from Sargassum thunbergii by human fecal inoculums, and its impact on human colonic microbiota. The results showed that ST-P2 was homogeneous with molecular weight of 48,788 Da, and consisted of arabinose, galactose, glucose, xylose, and mannose. The main linkage types were identified as (1 → 5)-α-L-Araf, (1 → 3)-α-L-Manp, (1 → 3,6)-β-D-Galp, (1 → 6)-α-D-Glcp, and (1 → 3)-β-D-Xylp, respectively. After 48 h fermentation, 67.83 ± 1.15% of total carbohydrate was utilized by colonic microbiota. The pH value in the fecal culture significantly decreased from 6.09 ± 0.11 to 4.70 ± 0.04. The concentrations of total short chain fatty acids, acetic, propionic, n-butyric and n-valeric acids significantly increased compared to the blank. ST-P2 could remarkably modulate the composition and abundance of beneficial microbiota. These results suggest that ST-P2 could potentially be a functional food aimed at promoting the gut health.