Abstract: This experiment was conducted to investigate the effect of yeast β-glucan and bacitracin zinc on growth performance and intestinal microflora in earlyweaning calves. Twenty healthy Holstein male neonatal calves were randomly allotted to four treatments with five replicates each. The calves were fed with the diets supplemented with 0 (Treatment A), 75 mg·kg^-1 yeast β-glucan (Treatment B and C), 60 mg·kg^-1 bacitracin zinc (Treatment D). The experiment lasted for 28 days. On d 21, the calves of Treatments A, B and D were challenged orally with Escherichia coli (O141：K99), and calves of Treatment C were fed normally. Feed intake (FI) was recorded daily, body weight was measured and average daily gain (ADG) was calculated every two weeks, and fecal samples were collected from superior ampullar mucosa of the rectum to determine the intestinal microflora. The results showed as follows: Compared with Treatment A, the ADG of calves in Treatment B increased by 26.18% and 24.93% in the two phases before the Escherichia coli challenged (P<0.05), the ADG of calves in Treatment B and D increased by 30.38% and 30.82% after the Escherichia coli challenged (P<0.05). As for the F/G, which in Treatment B and D were significantly lower than that in Treatment A (P<0.05). The amount of Escherichia coli in rectum was rapidly increased after the challenged, compared with Treatment A, the amount of Escherichia coli in rectum at 12 and 24 h in Treatment B and D were significantly decreased (P<0.05), and the amount of Lactobacillus was significantly decreased in the Treatment D (P<0.05). PCR-DGGE of 16S rDNA was used to investigate the similarity index, the band number in Treatment C was significant higher than that in Treatment A and D (P<0.05).The degree of similarities of treatments were range from 50% to 75%. According to the results, β-glucan could improve the growth of claves and adjust the structure of intestinal microflora, thus using β-glucan in calves feed may decrease the usage of antibiotics.