This study was to investigate the structure of a polysaccharide fraction from the Fortunella margarita and the relationship between its digestibility and structure. A novel polysaccharide fraction extracted by graded precipitation at ethanol concentrations of 20% from F. margarita (named FP20) comprised mainly glucose, galactose, and mannose. The unit composition was →4)-β-Glcp-(1 → 2)-α-Glcp-(1 → 2)-α-Galp-(1 → 4)-α-Galp-(1→ bone, and in →2)-α-Galp-(1→) with a branching point at C of β-Manp. FP20 was identified as a mannogalactoglucan with a different monosaccharide composition ratio and side-chain sugar residues compared with other plant polysaccharides. Moreover, FP20 had a spherical aggregations by atomic force microscope test. FP20 had an island-shaped structures with a smooth surface revealed by field emission scanning electron microscopy. Furthermore, in vitro digestive test, FP20 was resistance to a digestion system of saliva-gastric-small intestinal. The digestibility of FP20 was related to its backbone unit, structure and tight, uniform, and spherical chain conformation in aqueous.