Objective To explore the correlation between intestinal flora characteristics and diarrhea risk in patients with gastric cancer during chemotherapy. Method 120 patients with gastric cancer who underwent radical gastrectomy from August 2018 to February 2022 in the Department of Oncology of the First People's Hospital of Huai'an were selected as the research object. All patients received chemotherapy after operation and were divided into two groups according to whether diarrhea occurred during chemotherapy or not. The intestinal flora was determined by 16S rDNA gene sequencing and the characteristics composition and differences of intestinal flora were compared between the two groups. The related factors affecting diarrhea were analyzed by multivariate Logistic regression. Draw receiver operating characteristic curve ROC and analyze the characteristics of intestinal flora through the area under the curve AUC to guide the chemotherapy diarrhea. Result 16S rDNA gene sequencing was used to analyze 120 fecal flora samples Shannon's index 2. 92 ± 0. 43 Simpson 's index 1. 71 ± 0. 63 Chao1 's index 781. 26 ± 124. 26 and Ace index 7635. 26±225. 74 in the occurrence group and those in the non-occurrence group 2. 38±0. 61 、 1. 95±0. 72 、 715. 26±147. 25 、 7706. 68±2534. 68 . There was no significant difference P>0. 05 . The Beta diversity in the occurrence group 0. 31±0. 10 was lower than that in the non-occurrence group 0. 46±0. 13 P < 0. 05 . The sequence of patients' fecal flora mainly belongs to four phyla including Proteobacteria Firmicutes Bacteroidetes and Proteobacteria. In the occurrence group Actinomycetes 5. 56±1. 05 Firmicutes 69. 64±2. 06 Bacteroides 16. 75±2. 19 Proteobacteria 18. 26±2. 74 and non-occurrence groups 4. 42±0. 74 72. 65±1. 55 . Multivariate Logistic regression results showed that Beta diversity OR= 1. 320 95%CI = 1. 110-1. 570 was the risk factors for diarrhea Bacteroides OR = 0. 761 95%CI = 0. 629-0. 921 and Actinomycetes OR= 0. 255 95%CI = 0. 120-0. 521 were a protective factor in the occurrence of diarrhea. Conclusion For patients with gastric cancer receiving chemotherapy the change and composition of intestinal flora are the related factors of diarrhea during chemotherapy so we can give targeted medication guidance through the change of intestinal flora.