Mulberry is a kind of crop with high value and worldwide distribution. Mulberry leaf is being an excellent source of phytochemicals. In present study, a polysaccharide (MLP-2C) with an average Mw of 59.8 kDa was isolated and purified from mulberry leaf by hot water and chromatographic purification. Methylation, GC-MS and NMR analysis revealed that the main glycosidic linkage types of MLP-2C were (1→4)-α-D-GlcpA (28.91%), (1→2,6)-α-D-Glcp (26.81%) and (1→4)-α-D-Manp (29.64%) residues. The side chains mainly composed of (1→2)-α-D-Manp and (1→4)-β-D-GalpA residues which were terminated with α-D-Xylp residue. Within the tested concentrations (100–300 μg/mL), MLP-2C significantly inhibited cholesterol synthesis, promoted bile acid transport and cholesterol efflux by up-regulating the corresponding mRNA expression levels in HepG2 cells (P < 0.05). MLP-2C also exhibited high antioxidant capacity by scavenging of ROS and improving the activities of a series of antioxidant enzymes. The findings indicated that MLP-2C might be a good candidate for potential medicine or functional food on hypercholesterolaemia treatment.