Objective To initially explore the sequential changes in the intestinal flora of corpse for the estimation of postmortem interval （PMI）. Methods Rats were sacrificed by cervical dislocation, and samples were taken from their intestines using cotton swab to extract the DNA of intestinal flora. The 16S rRNA V3 universal primers were selected for PCR, and the PCR products were used for denatured gradient gel electrophoresis. The diversity and similarity analysis of intestinal flora were analyzed between groups, and the bands were cut from denaturing gradient gel electrophoresis. After purification, PCR and sequencing, the percentage of major bacteria in each group was obtained. Results The flora diversity showed a reduced tendency from 1st to 30th day after death （P<0.05）, while the intra-group similarity showed a downward trend （P<0.05）. The number of bands and intra-group similarity coefficient （Cs） on the first day was higher than that of other groups （P<0.05）. The intra-group Cs of the 25th and 30th day had a significant difference compared with the 5th day （P<0.05）. At the genus level, the intestinal flora was mainly composed of Enterococcus sp. on the 1th and 5th day after death, Bacillus thuringienssis was the dominant species on the 10th, 15th and 20th day, and Enterococcus faecalis became the dominant species on the 25th and 30th day. Conclusion The composition and structure of intestinal flora change significantly in rats with the time after death, which indicates that the succession of intestinal flora is related to the postmortem interval.