The purpose of this study was to investigate the effects of alanyl-glutamine (Ala-Gln) and mannooligosaccharide (MOS) on intestinal morphology and microflora structure of piglets. A total of 162 piglets weaned at 21 days of age were randomly allotted to 9 groups with 3 replicates per group and 6 piglets per replicate. The control group was fed a basal diet, and the other groups were fed the basal diet supplemented with 0.10% MOS, 0.20% MOS, 0.15% Ala-Gln, 0.30% Ala-Gln, 0.10% MOS+0.15% Ala-Gln, 0.20% MOS+0.15% Ala-Gln, 0.10% MOS+0.30% Ala-Gln and 0.20% MOS+0.30% Ala-Gln, respectively. On the 14th and 28th day of the experiment, one piglet was randomly selected from each repeat for slaughter, small intestine tissue samples were collected, intestinal villi ultrastructure was observed by transmission electron microscopy, and microbial diversity was statistically analyzed. The results showed as follows: 1) compared with the control group, the villi height of duodenum, jejunum and ileum and the villus height/crypt depth (V/C) of duodenum and ileum were significantly increased after adding 0.10% MOS+0.30% Ala-Gln and 0.20% MOS+0.30% into the diet (P<0.05), and the micro-villi were clear, orderly and dense on the whole. 2) Compared with the control group,dietary 0.20% MOS+0.15% Ala-Gln was increased the relative abundance of beneficial bacteria such as Lactobacillus, Coprococcus and Fibrobacteriaceae in piglets (P<0.05). 3) MOS and Ala-Gln had significant interaction effects on jejunal villi height of piglets at 35 days of age, duodenal villi height and duodenal V/C of piglets at 49 days of age (P<0.05). Therefore, the combination of Ala-Gln and MOS can regulate the growth and development of intestinal villi in weaned piglets, improve the microflora structure of cecum, and increase the relative abundance of beneficial bacteria, thus promoting the intestinal health of piglets.