The probiotic strain Bacillus amyloliquefaciens JM033 (B. amyloliquefaciens JM033), isolated from the traditional Chinese fermented food Sufu (also known as Fu-ru or fermented bean curd), is distinguished by its high production of exopolysaccharides (EPS). The EPS (BAP-1) produced by this strain was purified and its structure analyzed. BAP-1 is a novel hybrid fructan with a molecular weight of 17.6 kDa. It is composed of →6)-β-D-Fruf-(2 → and →1,6)-β-D-Fruf-(2→, which form the backbone, with a branched chain of β-D-Fruf-(2 → attached at the 1-position of residue B. In vivo studies on mice indicated that BAP-1 improves immunity in immunosuppressed mice by enhancing humoral immunity (P <
0.01), monocyte-macrophage phagocytosis (P <
0.01), and NK cell killing activity (P <
0.05). Additionally, BAP-1 was found to improve the composition of the intestinal microbiota and stimulate the production of short-chain fatty acids. Notably, BAP-1 exhibited a significant effect on the proliferation of Akkermansia. Therefore, BAP-1 shows promise as a prebiotic and may contribute to the development of new immunomodulatory agents.