This study investigated the effects of substitution of whole corn silage (CS) with Broussonetia papyrifera silage (BPS) in different ratios on the serum indicators, hindgut fermentation parameters (pH, ammoniacal nitrogen, and volatile fatty acids), and fecal bacterial community of Holstein heifers. Sixteen heifers (8-month-old, 220±30 kg) were randomly divided into four treatments according to different BPS substitution ratios of feed basis (0%, 25%, 50%, and 75%). The experiment consisted of a 7-day preliminary feeding period and a 30-day experimental period. On the last day of the trial, the blood samples were collected from caudal vein, and the feces samples were collected from rectum. With the increasing of BPS content, the concentration of malondialdehyde (MDA) and interleukin-1β (IL-1β) in serum decreased (P<0.05), and the immunoglobulin A (IgA) and IL-4 content of serum increased (P<0.05); and the hindgut pH value increased (P<0.05). 16S rDNA sequencing found that the dominant phyla were Firmicutes, Bacteroidetes, and Verrucomicrobia; and the dominant genera were Ruminococcaceae_UCG-005, Ruminococcaceae_UCG-010, and Rikenellaceae_RC9_gut_group. Linear Discriminant Analysis Effect Size (LEfSe) analysis found 12 differential operational taxonomic units (OTUs) which have strong correlation with some serum and hindgut indicators. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) found that BPS have impacts on the pathways, such as carbohydrate transport and metabolism, and promotes amino acid transport and metabolism. To sum up, inclusion of BPS in heifer diets can affect the fecal bacterial community, and further improve serum anti-oxidant and immune indicators in Holstein heifers.