Background Disturbance of the intestinal flora is a pathogenic factor for chronic atrophic gastritis (CAG). Hua-Zhuo-Jie-Du (HZJD) has been shown to be an effective Chinese herbal preparation for treating CAG. However, the effects of HZJD on the intestinal flora of CAG is unclear. In this study, we probed the regulating effects of HZJD on intestinal microbes in CAG rats using 16S rRNA gene sequencing. Methods High-performance liquid chromatography (HPLC) analysis was used to perform quality control of HZJD preparations. We then administered 1-methyl-3-nitro-1-nitrosoguanidine (200 mu g/ml) to Sprague-Dawley rats to establish a CAG model. HZJD and vitacoenzyme were administered orally to these rats over a 10 week period. Hematoxylin and eosin (H&E) staining was performed to observe the histopathology of CAG rats. A rarefaction curve, species accumulation curve, Chao1 index, and ACE index were calculated to assess the alpha diversity. Principal component analysis (PCA), non-metric multi-dimensional scaling (NMDS), and unweighted pair group method with arithmetic mean (UPGMA) were conducted to examine the beta diversity. The LEfSe method was used to identify differential bacteria. Differential function analysis used PCA based on KEGG function prediction. Results HPLC showed that our HZJD preparation method was feasible. H&E staining showed that HZJD significantly improved the pathological state of the gastric mucosa in CAG rats. The rarefaction curve and species accumulation curve showed that the sequencing data were reasonable. The Chao1 and ACE indices were significantly increased in CAG rats compared to the N group. Following HZJD and vitacoenzyme treatment, the Chao1 and ACE indices were decreased. PCA, NMDS, and UPGMA results showed that the M group was separated from the N, HZJD, and V groups, and LEfSe results showed that the relative abundance of Akkermansia, Oscillospira, Prevotella, and CF231 were significantly higher in the N group. Proteobacteria and Escherichia were significantly enriched in the M group, Allobaculum, Bacteroides, Jeotgalicoccus, Corynebacterium, and Sporosarcina were significantly enriched in the V group, and Firmicutes, Lactobacillus, and Turicibacter were significantly enriched in the HZJD group. Conclusion HZJD exhibited a therapeutic effect on the intestinal flora of CAG rats.