Polysaccharides are key bioactive compounds in lotus plumule tea, but their anti-diabetes activities remain unclear. The purpose of this study was to investigate the prebiotic activities of a novel polysaccharide fraction from the Nelumbo nucifera lotus plumule, and to examine its regulation of glucose metabolism in insulin-resistant HepG2 cells. The N. nucifera polysaccharide (NNP) was purified after discoloration, hot water extraction, ethanol precipitation, and DEAE-cellulose chromatography to obtain purified polysaccharide fractions (NNP-2). Fourier transform infrared spectroscopy was used to analyze the main structural characteristics and functional group of NNP-2. Physicochemical characterization indicated that NNP-2 had a molecular weight of 110.47 kDa and consisted of xylose, glucose, fructose, galactose, and fucose in a molar ratio of 33.4:25.7:22.0:10.5:8.1. The prebiotic activity of NNP-2 was demonstrated in vitro using Lactobacillus and Bifidobacterium. Furthermore, NNP-2 showed bioactivity against α-glucosidase (IC50 = 97.32 µg/mL). High glucose-induced insulin-resistant HepG2 cells were used to study the effect of NNP-2 on glucose consumption, and the molecular mechanism of the insulin transduction pathway was studied using RT-qPCR. NNP-2 could improve insulin resistance by modulating the IRS1/PI3K/Akt pathway in insulin-resistant HepG2 cells. Our data demonstrated that the Nelumbo nucifera polysaccharides are potential sources for nutraceuticals, and we propose functional food developments from the bioactive polysaccharides of N. nucifera for the management of diabetes.