Objective To investigate the effect of chemotherapy on the intestinal flora and signaling pathways in patients with primary lung cancer. Methods 26 patients with primary lung cancer who received chemotherapy for the first time from February 2019 to July 2019 were enrolled, and fresh stool samples were collected within 48 to 72 hours before chemotherapy (CBG group) and after the second cycle of chemotherapy (CAG group). The Illumina MiSeq high⁃throughput sequencing platform was used to sequence the 16S rRNA genes of gut microbes in fecal specimens. QIIME 2.0 was used to process the sequencing data and to predict the functional characteristics of the gut microbiota through PICRUST. Results Compared with the CAG group, the CBG group had higher intestinal bacteria richness, but the difference in the diversity of the two groups of bacteria was not statistically significant (P>0.05), in terms of the composition of intestinal flora, the CBG and CAG groups had four common phylum of superior bacteria includes Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria(P<0.05). The genus composition of CBG group Faecalibacterium, Oscillospira and Alipipes were significantly reduced, while Veillonella, Streptococcus and f_Christensenellaceae_uncl. were significantly increased. In terms of functional metabolism, it was found that the metabolic type of the intestinal microbiota in the CAG group was up⁃regulated in fatty acid metabolism, tryptophan metabolism, glycolysis and other functions. Conclusion After the first two cycles of chemotherapy for patients with primary lung cancer, the pathogenic bacteria in the intestinal condition increased while the butyrate⁃producing bacteria decreased. The tumor⁃promoting, drug⁃resistant metabolic pathways and inflammation⁃related metabolic pathways were up⁃regulated. It is suggested that the damage of the intestinal mucosa is not easy to recover, and the function of intestinal barrier is decreased.