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The objective of this study was to investigate the structural characterization of a polysaccharide fraction from Fortunella margarita and its molecular structural change under simulating digestion in vitro. The result showed that the polysaccharide fraction prepared and purified by graded precipitation (named as FP40) was characterized as a novel galactoglucan with unit composition having a backbone structure of → 4)-β-Glcp-(1 → 4)-α-Glcp-(1 → 4)-α-Galp-(1 → and a side chain of β-Glcp-(1 → 4)-β-Manp-(1 → 6)-β-Glcp. FP40 displayed a random-coil chain conformation and a “thin-slice” shape observed by atomic force microscope and scanning electron microscope. Moreover, the saliva had no effect on the molecular weight of this galactoglucan, but it could change its chain conformation. In contrast, FP40 could be digested by gastric and small-intestinal juices. After small-intestinal digestion, the chain conformation of FP40 changed from random coil to sphere conformation. Compared with mannogalactoglucan from Fortunella margarita, it exhibited different unit composition, chain conformation, surface morphology, and digestibility.