Abstract: The extraction, isolation, purification, and structural characterization of millet bran polysaccharides (MBP), as well as its antibacterial activity were conducted to provide a reference for the development and utilization of nutrients in the processing byproducts of millet. An alkali extraction and alcohol precipitation method was used to extract the polysaccharides. Subsequent purification was achieved through column chromatography utilizing DEAE-50 and Sephadex G-100. Structural analysis of the millet bran polysaccharides was performed using a combination of techniques, including HPLC chromatography, infrared spectroscopy, atomic force microscopy, scanning electron microscopy, and X-ray diffraction. The results showed that the molecular weight of MBP was determined to be 3.479×10⁴ Da. The monosaccharide composition was exhibited a molar ratio of mannose:rhamnose:glucose:galactose:arabinose:xylose=0.11:0.13:5.86:0.62:1.00:0.52. Moreover, MBP was identified as a β-type pyranose polysaccharide and exhibits remarkable thermal stability. In terms of morphology, MBP was laminated, closely connected, and had no crystal structure. The antibacterial assay unveiled the minimum inhibitory concentration of MBP against Staphylococcus aureus was 2 mg/mL. This study showed that the polysaccharides from proso millet bran had good antibacterial ability.