Antibiotics are commonly provided to weaned piglets; however, this practice has become controversial due to the increased occurrences of microbial resistance, and alternatives are needed. This study aimed to investigate the effects of dietary supplementation with yeast glycoprotein (YG) on growth performance, intestinal mucosal morphology, immune response and colonic microbiota in weaned piglets. A total of 240 weaned piglets (d 23 ± 2) from 16 pens (15 piglets per pen) were randomly allocated to an antibiotics group (25% quinocetone 200 mg kg-1 and 4% enduracidin 800 mg kg-1 of the basal diet) or a YG group (800 mg kg-1 YG of the basal diet), respectively. The trial lasted 14 days, and at the end of the trial, one piglet per pen was chosen to collect plasma, intestinal tissue and colonic digesta samples. The results indicate that piglets fed diets containing YG tended to show increased final body weight (0.05 < P < 0.1), increased average daily gain (P < 0.05) and decreased F/G (P < 0.05) when compared with the antibiotics group. Moreover, intestinal permeability showed that YG led to an improvement in the intestinal development via decreasing serum content of DAO (P < 0.01). Histological evaluations showed that YG contributed to the improvement of the intestinal development via increasing villous height (P < 0.05) and the villous height to crypt depth ratio (P < 0.01), and decreasing crypt depth (P < 0.01) and villous width (P < 0.05) in the ileum. Intestinal integrity also showed that YG was conducive to improvement of the intestinal development via upregulating the m-RNA expression of occludin (P < 0.05) in the duodenal and jejunal mucosa. Interestingly, YG supplementation downregulated the m-RNA expression of IL-12 (P < 0.05), upregulated the m-RNA expression of Hsp-70 (P < 0.05) in the duodenal mucosa, downregulated the m-RNA expression of Hsp-70 (P < 0.05) and IFN-γ (P < 0.05), upregulated the m-RNA expression of Hsp-90 (P < 0.05) in the jejunal mucosa, and upregulated the m-RNA expression of Hsp-70 (P < 0.05) in the ileal mucosa. On the other hand, colonic microbiota results showed that YG supplementation increased the relative abundance of Lactobacillus (P < 0.05) in the genus level. Colonic metabolite results showed that YG supplementation decreased the content of acetate (P < 0.05). Taken together, it is speculated that YG would be a potent alternative to prophylactic antibiotics in improving the gut health in weaned piglets.