Objective To analyze the gut microbiota of patients with chronic hepatitis B (CHB) with acute exacerbation and healthy people with 16S ribosomal RNA (16S rRNA) amplification sequence technology, and to explore the influence of gut microbiota on these patients and its possible mechanism. Methods A case-control trial was conducted on 20 patients with acute exacerbation of CHB (CHB group) admitted to Department of Infectious Diseases and 23 healthy controls (control group) in Department of Physical Examination from the Second Affiliated Hospital of Air Force Military Medical University from April to September 2021. Their fecal samples and clinical data were collected. 16S rRNA high-throughput sequencing was used to sequence the V3-V4 gene amplification products of the gut microbiota of the participants. The “t test” function in R language was used for independent sample t test to analyze the α diversity differences in the gut microbiota between the 2 groups. The “ade4” package in R language was employed for principal coordinates analysis (PCoA) based on the weighted Unifrac distance in order to compare the β diversity differences in gut microbiota between the two groups. The “t test” function in R language was conducted for independent sample t test to analyze the differences in gut microbiota between the two groups at the levels of phylum, family and genus. PICRUST2 software was adopted to predict and analyze the metabolic pathways of gut microbiota. Results Compared with the control group, α diversity analysis showed that the fecal microbial diversity and abundance were decreased in the CHB patients with acute exacerbation (P<0.001). β diversity analysis indicated that the flora communities of the two groups were clustered separately, with significant differences (F=4.931, P=0.001). At the portal level, the relative abundance of Firmicutes in the flora of the CHB patients with acute exacerbation was obviously lower (P<0.01), while that of Actinobacteria was statistically increased (P<0.001) than that of control group. At the family level, the relative abundance of Porphyromonadaceae, Rikenellaceae, Selenomonadaceae, and Ruminococcaceaede was notably lower (P<0.001), while that of Streptococcaceae was remarkably increased (P<0.05) in the patients than the control group. At the genus level, the relative abundance of Alistipes, Bacteroides, Blautia, Clostridium, Dialister, Faecalibacterium, Gemmiger, Kineothrix, Megamonas, Parabacteroides and Ruminococcus was significantly decreased (P<0.05), while that of Prevotella and Veillonella was higher (P<0.05) in the patients than the control group. The prediction analysis of intestinal flora function revealed that compared with control group, the pathways related to polysaccharide biosynthesis and metabolism and lipid metabolism were more abundant in the CHB patients with acute exacerbation. Conclusion The diversity and richness of gut microbiota in CHB patients with acute exacerbation are significantly lower than those in healthy people, and their community structure has changed significantly. The imbalance of gut microbiota may be an important cause of acute exacerbation of CHB.