Garlic saccharides have prebiotic activity, but the association between their function and structure is still poorly known. In present study, four different garlic saccharides were obtained from garlic polysaccharides (GPs) after acidolysis by ultrafiltration. Obtained GPs were constituted by different monosaccharides, among which fructose and glucose were the main components, while galactose was a major component of GPs-U6. All four saccharides were partly degraded by the simulated digestive system, and most could reach the large intestine to be utilized by the gut microbiota. Except for GPs-U6, the other three garlic saccharide fractions had good prebiotic activity in vitro and in vivo. Furthermore, GPs-U0.3 with lower molecular weight (Mw) showed better prebiotic activity, including promoting the production of short-chain fatty acids (SCFAs), increasing the abundance of beneficial bacteria such as Bifidobacterium, Lachnospiraceae NK4A136 group and Phoscolarctobacterium, and inhibiting the growth of potentially harmful bacteria. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis showed that GPs-U0.3 could reduce the risk of cancer and cardiovascular diseases. Overall, this findings of the present study revealed the digestive properties of GPs, as well as the potential association between their chemical structures and fermentation characteristics by gut microbiota. Thus, it can be stated that GPs-U0.3 can be used as potential prebiotics in functional foods, which provides a theoretical basis for the targeted preparation of functionalized garlic saccharides.