Recently, several studies have indicated that the intestinal microbiota can be regulated by the individual attributes, and even the alternation of circadian rhythm. Inspired by this, we speculated that seasonal variation might also have some effect on the intestinal microbiota. A total of 11 Sprague-Dawley male rats, weighing 250-280 g, were divided into summer group (n = 5) and winter group (n = 6). Cages were individually ventilated at 20 ± 2 °C and 45-65% relative humidity with a circadian rhythm of 12/12 h. After 1 week of adaptively feeding, mucosal contents of jejunum, terminal ileum, and ascending colon were collected and analyzed by 16S rRNA Gene Amplicon Pyrosequencing. The results showed that intestinal microbiota of rats for the same strain were affected by season change under the same feeding condition and living environment. We should take seasonal factor into account in the future experimental design based on intestinal microbiome.