Abstract: Channel catfish (Ictalurus punctatus) is an important species cultured in saline-alkali water in China. Salinity is a key environmental factor, but the mechanism of its influence on the intestinal flora of I. punctatus is not clear. To investigate theeffect of salinity on the intestinal flora of I. punctatus, we designed an eight-week experiment and divided the fish into four groups: control group (S0, salinity 0), low-salt group (S3, salinity 3‰), medium-salt group (S6, salinity 6‰), and high-salt group (S9, salinity 9‰). The intestinal contents of fish were collected every two weeks, and the composition and function of the intestinal flora were analyzed by 16S rRNA sequencing. The results show that with the increase of salinity, the richness of theintestinal flora increased, but the diversity decreased significantly. On phylum level, Proteobacteria, Firmicutes, Fusobacteriota, Bacteroidetes, Cyanobacteria and Actinobacteria were the dominant bacterial phyla. In the control, low-salt and medium-salt groups, the intestinal flora showed rhythmic changes in the 2-week cycle, regularly repeating between Proteobacteria and Firmicutes, Fusobacteriota, Bacteroidetes. In contrast, the high salinity group disrupted the rhythm of changes, showing a maintenance of the predominance of Proteobacteria. Meanwhile, the functions of the intestinal flora also changed significantly in high-salinity condition, with significantly higher functional abundance in fatty acid metabolism and degradation, vitamin and amino acids degradation, and energy metabolism, and a significant decrease in the genetic repair functions such as mismatchrepair, nucleotide excision repair. High salinity caused strong stress in I. punctatus, increased energy expenditure, and ultimately altered the composition and function of the intestinal flora. Therefore, it is recommended to culture I. punctatus in saline ponds, which requires higher energy and probiotic supplementation in feed.