Abstract: Objective To investigate the characteristics of intestinal microbiota and its relationship with early-onset sepsis (EOS) in premature infants with premature rupture of membranes(PPROM), so as to provide evidence for early diagnosis and treatment of EOS. Methods A total of 38 PPROM infants who were admitted to Department of Neonatology, the Affiliated Hospital of Xuzhou Medical University from March 2019 to September 2020 were enrolled. According to EOS diagnostic criteria, they were divided into two groups: an EOS group (n=20) and a non-EOS group (n=18). Meanwhile, 10 normal premature infants were selected as a control group. High-throughput sequencing was used to analyze the differences of bacteria in the fecal samples of preterm infants in each group one day after birth. Results For diversity analysis, no statistical difference was found in the Chao1 indexes of fecal flora among the three groups,while the Shannon index was significantly lower in the EOS group than those of the non-EOS group and control group (P<0.05). On the phylum level, there was no statistical difference in the relative abundance of Firmicutes, Proteobacteria, and Bacteroidetes among the three groups (P<0.05), while the relative abundance of Actinobacteria in the EOS group was lower than that in the control group (P<0.05). On the Genus level, the EOS group presented increases in the relative abundance of Klebsiella, and decreases in Enterococcus, compared with the non-EOS group and the control group; the relative abundances of Clostridium, Lactobacillus and Bifidobacterium in the EOS group were lower than that in the control group; the relative abundances of Ureaplasma and Streptococcus in the non-EOS group were higher than that in the control group (P<0.05). The pathogenic bacteria identified in the intestinal microbiota from the five children with positive blood culture were all the bacteria with the highest relative abundance. Conclusions The diversity of intestinal microbiota in premature infants with PPROM decreases, but the relative abundance of opportunistic pathogens such as Klebsiella and Staphylococcus increases. The decreases in the relative abundance of beneficial bacteria may be related to the occurrence of EOS.