In this work,the impact of high fat diet on gut microbiota in the male and female C57BL/6J mice were investigated. Sixteen 8-week-old C57BL/6J mice(8 per sex)were housed in the specific pathogen free(SPF)condition. The mice fed with the normal diet were used as the control groups,fed with the 45% kcal fat diet were used as the experimental groups(4 mice in each group). After 6 weeks of dietary intervention,fresh fecal samples were collected from each mouse. For high throughput HiSeq 2500 sequencing analysis,fecal bacterial genomic DNA was extracted and amplified based on the V3+V4 region of the bacterial 16S-rDNA gene by PCR(polymerase chain reaction). The changes in composition and relative abundance of gut microbiota were evaluated. The results showed that high-fat diet altered gut microbiota composition,affecting the diversity and similarity. In the female high-fat diet group,the relative abundance of Proteobacteria phylum significantly decreased(P<0.05)and the Actinobacteria phylum significantly increased(P<0.01)compared with the control diet group. However,in the male high-fat diet group,the relative abundance of Proteobacteria phylum significantly increased(P<0.01)and the Actinobacteria phylum had no obvious change compared with the control diet group. Moreover,the relative abundance of L.murinus was significantly higher in the high-fat diet group than that in the control diet group of female mice(P<0.01). However,this species of bacteria did not show significant change in the male high-fat diet group. The effects of high-fat diet on the composition and structure of gut microbiota were different in the female and male mice,these results provide data for further studying the effects of dietary and gender factors on intestinal microorganisms.