The development of novel prebiotics, which could regulate the intestinal microbiota, may help prevent and treat intestinal diseases. Here, we studied a homogeneous polysaccharide, LPE-2, produced by Lactobacillus pentosus YY-112 during fermentation. Methylation and gas chromatography-mass spectrometry analysis, combined with nuclear magnetic resonance results, suggested that the structural unit of LPE-2 comprises a branched mannan moiety and a linear glucan moiety. In vitro simulated intestinal fermentation showed that LPE-2 reduced harmful intestinal gas production and promoted short-chain fatty acid production (especially propionic acid). Moreover, it reduced the relative abundance of Escherichia-Shigella, increased that of Bifidobacterium and Lactobacillus, and had a stronger regulatory effect on intestinal flora in women than in men. The potential sex-specific prebiotic effects of LPE-2 on human intestinal health, were possibly related to its mannan branch with (1→2) and (1→3) linkages and backbones with flexible α configurations, which are sheared and degraded/utilized easier by Bifidobacterium and Lactobacillus.