Abstract: Six litters (8-11 piglets per litter) of healthy neonatal crossed-bred piglets (Duroc×Landrace×Yorkshire) were randomly divided into a control group and treatment group. Each group was composed of three litters, comprising triplicates. The animals in the control group were offered regular starter feed, while those in the treatment group were fed starter feed with 1% replacement of fructo-oligosaccharides (FOS) (by dry matter %) at 7 days after birth. The experiment was ended 23 days (weaning) after birth. At 7 and 23 days, one piglet from each triplicate was slaughtered. Pancreas, gastrointestinal tract (GIT) tissues and digesta were collected to investigate effects of FOS on mucosal morphology, digestive enzyme activity, organic acid and Lactobacillus of the GIT of unweaned piglets. The results showed that the villi height and the ratio of villus height and crypt depth were increased by FOS treatment in the jejunal and ileum (P<0.05). The highest amylase activity was in the pancreas and duodenum of the FOS group (P<0.05). Lipase and protease activity were not significantly higher in the FOS group than in the control group (P>0.05). Higher VFAs (volatile fatty acids) concentrations in the GIT and lower lactic acid concentrations in the stomach were observed in the FOS treatment (P<0.05). The number, similarity and diversity of Lactobacillus communities were also increased by FOS supplementation. The findings imply that the small intestinal morphological development of unweaned piglets, the activities of digestive enzymes, and VFAs concentration in gastrointestinal contents were improved by FOS addition. FOS can be utilized by some species of Lactobacillus, which enhances the stability of microbial community structure in the GIT of unweaned piglets, resulting in a beneficial contribution to the enteric health of piglets and a possible smooth transition at weaning.