Twelve healthy weaned piglets with similar body weight were randomly divided into two groups, the control group(CON group) and the experimental group(SDGS group), and fed with an isonitrogenous isoenergetic basal diet formulated according to the NRC(2012) nutritional requirements standard for piglets and the 4% distiller’s grains Saccharomyces cerevisiae culture-supplemented diet, respectively, with six replicates in each group, the last time of the experiment was conducted for 28 days after 7 days of pre-feeding. The effects of dietary supplementation of Saccharomyces cerevisiae culture containing distiller’s grains on growth performance, organ index, serum biochemical indexes, intestinal morphology, and relative mRNA expression levels of intestinal mucosa tight junction(TJ) proteins and cytokines of weaned piglets were collected to comprehensively evaluate the feeding effects of dietary supplementation of Saccharomyces cerevisiae culture containing distiller’s grains on weaned piglets. The results showed that the average daily gain weight of the SDGS group was significant(P<0.05) higher than that of the CON group, and the average daily feed intake and feed/weight ratio had no significant(P>0.05) changes. The liver index of the SDGS group was significantly(P<0.05) lower than that of the CON group, while the organ indexes of spleen, heart, pancreas and kidney showed no significant(P>0.05) changes. The differences of serum biochemical indexes(total protein, urea, glucose, immunoglobulin M and immunoglobulin G) between the two groups were not statistically significant(P>0.05). Compared with CON group, the intestinal morphology of the SDGS group was more complete and aligned, but the jejunal and ileal crypt depth were significantly(P<0.05) increased, and the jejunal villus height was significantly(P<0.05) decreased. The ratios of jejunal and ileal villus height to crypt depth were all significantly(P<0.05) decreased, while the duodenal crypt depth showed an increasing trend(P=0.081), but the duodenal villus height, crypt depth and the ratio of villus height to crypt depth showed no significant(P>0.05) changes. In the SDGS group, the mRNA relative expression levels of Claudin-3 in the TJ protein of jejunal mucosa were decreased(P=0.091), while the relative mRNA expression levels of closed small loop protein-1(ZO-1) and occludin in the jejunal mucosa TJ protein and the mRNA expression levels of Claudin-3, occludin and ZO-1 in the duodenal and ileal mucosa TJ proteins were not significantly changed(P>0.05). In the SDGS group, the mRNA relative expression levels of cytokine IL-6(IL-6) in duodenal and ileal mucosa were significantly(P<0.05) decreased, while the mRNA relative expression level of cytokine IL-10 in ileal mucosa was decreased(P=0.068), the mRNA relative expression levels of cytokine IL-1β(IL-1β), IL-10, IL-6, IL-1β, IL-10 and IL-1β in jejunum and IL-1β in ileal mucosa were not significant(P>0.05). In conclusion, according to the NRC(2012) nutritional requirements standard for piglets, the isonitrogenous isoenergetic diet prepared with the addition of 4% distiller’s grains Saccharomyces cerevisiae culture could significantly improve the average daily gain weight of weaned piglets, significantly reduce liver index, improve intestinal immune function without the intestinal barrier structure damage and showing more complete intestinal mucosa structure, while the intestinal morphology development showed a trend towards the reduction of digestion and absorption capacity. It could be seen that the Saccharomyces cerevisiae culture from distiller’s grains could be used as the raw material for the diets of weaned piglets, but attention should be paid to the protection of intestinal health during its usage.