A total of 180 weanling pigs (21 ± 3 d of age; 5.98 ± 0.04 kg) were used to investigate the effect of chito-oligosaccharide (COS) on growth performance, intestinal barrier function, intestinal morphology, and cecal microflora. Based on initial BW, gender and litter, the pigs were given 5 treatments during a 14-d feeding experiment, including a basal diet (control), 3 diets with COS supplementation (200, 400, or 600 mg/kg), and a diet with colistin sulfate (CSE) supplementation (20 mg/kg). Six randomly selected pigs from each treatment were used to collect serum, duodenal, jejunal, ileal, and cecal samples on d 7 and 14 postweaning. From d 1 to 7 postweaning, pigs fed COS or CSE had greater ADG and ADFI compared with the control pigs. From d 1 to 14, diets with either 400 or 600 mg/kg COS, or 20 mg/kg CSE increased (P < 0.05) ADG and G:F compared with the control diet. No significant differences were observed in ADG, ADFI, and G:F between the pigs fed COS and CSE. Pigs fed either 400 or 600 mg/kg COS, or 20 mg/kg CSE had less (P < 0.05) diamine oxidase (DAO) in the serum, but greater concentration of (P < 0.05) DAO in jejunal mucosa, than the control pigs on d 7 postweaning. Treatments did not affect villous height and crypt depth of the duodenum, jejunum, or ileum. Pigs fed COS at 400 mg/kg had greater (P < 0.05) concentration of Bifidobacteria and Lactobacilli in the cecum than pigs fed the control diet and CSE diet on d 7 postweaning. Supplementation of COS or CSE decreased (P < 0.05) the population of cecal Staphylococcus aureus compared with the control diet on d 7 postweaning. The number of cecal Bifidobacteria in pigs fed 600 mg/kg COS was greater (P < 0.05) than that of pigs fed the control diet or CSE diet on d 14 postweaning. No significant differences were observed in Escherichia coli counts in the cecum among treatments. The present results indicate that dietary supplementation of COS at 400 or 600 mg/kg promotes growth performance and improves gut barrier function, increases the population of Bifidobacteria and Lactobacilli, and decreases S. aureus in the cecum of weanling pigs.