Vancomycin-resistant Enterococcus (VRE) caused nosocomial infections are rising globally. Multiple measures have been investigated to address this issue, altering gut microbiota through dietary intervention represents one of such effort. Stachyose can promote probiotic growth, which makes it a good candidate for potentially inhibiting VRE infection. This study aimed to determine whether stachyose inhibits VRE colonization and investigated the involvement of gut microbiota this effect of stachyose. In VRE-infection experiment, 6-week old female C57/6 J mice pre-treated with vancomycin were infected with 2 × 10 CFU VRE via gavage. These mice then received oral administration of stachyose or PBS as control for 7days. Two groups of uninfected mice were also received daily gavage of stachyose or PBS for 7 days to observe the impact of stachyose treatment on normal mice. Fresh fecal and colon samples were collected, then VRE colonization, gut microbiota and gene expression were respectively assessed using cultivation, 16s rRNA sequencing and RNA-sequencing in two parallel experiment, respectively. In VRE-infected mice, stachyose treatment significantly reduced VRE colonization on days 9 and 10 post-infection. Stachyose treatment increased the relative abundance of Porphyromonadaceae, Parabacteroides, and Parabacteroides distasonis compared to the PBS-treated infection mice (P < 0.01). Uninfected mice treated with stachyose showed a significant increase in Lactobacillaceae and Lactobacillus compared to the PBS-treated uninfected mice(P < 0.05). RNA-sequencing results showed that stachyose treatment in VRE-infected mice increased expression of genes involved in TNF and IL-17 signaling pathways. Stachyose treatment also up-regulated Hsd17b14, Cyp3a44, Arg1, and down-regulated Pnliprp2, Ces1c, Pla2g4c genes involving in metabolic pathway in uninfected mice. In conclusion, stachyose supplementation can effectively inhibit VRE colonization and probably altering composition of the microbiome, which can in turn result in changes in expression of genes. Stachyose may also benefit health by increasing the abundance of Lactobacillus and expression of genes involving in metabolic pathway in normal mice.