Objective To analyze the impact of carbapenem-resistant Enterobacterales (CRE) colonization on gut microbes in patients in intensive care unit (ICU) based on 16S rRNA sequencing. Methods Fresh stool or anal swab specimens from ICU patients were collected and inoculated on MacConkey plates containing 2 μg/mL ertape-nem for incubation. Strains were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility analysis was performed by disk diffusion method. 16S rDNA fragments of preliminarily screened CRE strains were amplified by polymerase chain reaction (PCR), and CRE strains were further determined using micro-broth dilution method. Finally, genomic DNA of fecal or anal swab specimens from CRE colonized and non-colonized patients was extracted for 16S rRNA sequencing analysis on the HiSeq platform. Results A total of 241 stool or anal swab specimens were collected from ICU patients, including 17 CRE-positive specimens. 726 OTUs were isolated by sequencing the V3-V4 region of the 16S amplicon, 631 in the non-colonized group and 480 in the colonized group, with a total of 385 OTUs in two groups. Most of these shared OTUs belonged to the Firmicutes, Proteobacteria, Bacteroidetes, Verrucomicrobia and Actinobacteria. At the class level, there was a significant difference in the relative abundance of γ-proteobacteria (W=193.000, P < 0.001, FDR=0.004) and Synergistia (W=37.500, P=0.001, FDR=0.018) before and after CRE colonization. At the genus level, Klebsiella pneumoniae (W=195.000, P < 0.001), Enterococcus saccharolyticus (W=153.000, P=0.038) and Campylobacter hominis (W=63.500, P=0.050) between the two groups had significant diffe-rences. No significant impact of intestinal CRE colonization on the KEGG functional pathway of gut microbiota in ICU patients was found. Conclusion CRE colonization can decrease the diversity of gut microbes in ICU patients. CRE colonization group showed significant differences in the abundance of Klebsiella pneumoniae, Enterococcus saccharolyticus and Campylobacter hominis compared to the non-colonization group.