Antibiotics contribute a lot to human beings and can kill bacteria effectively. However, more and more studies show that antibiotics can disturb the intestinal microbial community. It has been widely reported that oral antibiotics can reduce the diversity of intestinal microflora, but the effect of intramuscular injection on intestinal microflora is less studied. In this study, we sequenced the intestinal microflora of mice treated with tetracycline by 16SrRNA method, and found that intramuscular injection of tetracycline (TET) can also reduce the intestinal microbial richness of mice. In addition, the results showed that within a certain range (3 mg), with the increase of TET injection concentration, the wind of intestinal microflora in mice decreased significantly. When the injection concentration reached saturation, although the amount of TET injection was increased, the degree of intestinal flora affected was not increased. The results showed that the degree of diversity decrease was in direct proportion to the amount of tetracycline injection in the saturated concentration, but not positively related to the high amount of TET injection after exceeding the saturated concentration.