<p>The settlement levels of indigenous bacteria show circadian rhythms in various regions of the rat alimentary tract. Numerous bacteria colonize between the mucosal folds of the ascending colon in rodents; however, the rhythm of bacteria colonizing the ascending colon remains to be clarified. Therefore, we first aimed to examine the diurnal changes in bacteria colonizing in the rat ascending colon. The settlement levels of indigenous bacteria were significantly higher at zeitgeber time (ZT) 18 (dark phase) than at ZT6 (light phase) in the region encompassing the aggregated lymphoid tissue in the ascending colon (ALT-AC). The bacterial composition around the ALT-AC was dominated by the phylum <i>Firmicutes</i> and the family <i>Lachnospiraceae</i>, displaying notable distinctions from the compositions found in cecal contents and feces. The relative abundance of some bacterial species around the ALT-AC, such as <i>Mucispirillum schaedleri</i>, changed significantly between ZT6 and ZT18. Furthermore, we explored the effect of bacterial expansion on gene expression in the ALT-AC at ZT18 by administrating antibiotics for 1&#xa0;day to inhibit bacterial growth. The antibiotic-treated group exhibited significant downregulation of multiple genes, including those associated with cell proliferation (<i>Plk3</i>), differentiation into goblet cells (<i>Spdef</i>, <i>Atoh1</i>, <i>Bhlha15</i>), and Golgi organization (<i>Gorasp2</i>). These results suggested that indigenous bacteria around the rat ALT-AC undergo diurnal changes in both settlement levels, peaking at the dark phase, and bacterial composition. In addition, bacterial expansion during the dark phase can induce changes in the expression of diverse genes, including genes associated with goblet cell differentiation.</p>