Abstract: This study investigated the structural characteristics, bioactivity, and regulatory effects of polysaccharides extracted from jackfruit pulp using a pulsed ultrasound-assisted method (PUAE-JP) on gut microbiota. The polysaccharides were purified and characterized using Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and nanoparticle size analysis. Its bioactivities were evaluated using in vitro antioxidant assays, carbohydrate-metabolizing enzyme inhibition tests, and fecal fermentation models. The results revealed that PUAE-JP is an amorphous acidic heteropolysaccharide primarily composed of glucose and galacturonic acid, with a loose porous morphology and strong surface electronegativity. In vitro activity assays revealed significant dose-dependent antioxidant capacity (inhibitory concentration IC₅₀ values of DPPH, ABTS, and hydroxyl radicals were 3.23, 3.08, and 3.70 mg/mL, respectively) and significant inhibitory effects on α-glucosidase and α-amylase (IC₅₀ values of 4.63 and 3.07 mg/mL, respectively), with a non-competitive dominant mixed inhibition mechanism. Furthermore, in vitro fermentation experiments showed that PUAE-JP modulated the gut microbiota structure by enriching beneficial genera such as Megamonas and Megasphaera, while suppressing potential pathogens such as Fusobacterium. In addition, it promotes the production of short-chain fatty acids (SCFA) and upregulates metabolic pathways related to carbohydrate utilization. In conclusion, PUAE-JP exhibited promising hypoglycemic activity and probiotic potential, making it a functional food ingredient that requires further development.