Four small canines, suffered from pyometra, were employed to test the effects of uterus extirpation on the microbial community structure in their vagina and feces. The recycled common and specific bands derived from samples in vagina and feces were sequenced and cloned by adopting polymerase chain reaction and denaturing gradient gel electrophoresis (PCR–DGGE) technologies. The results showed that the diversity index, evenness and richness of flora in vagina were 3.22, 3.35 and 3.05, respectively before the extirpation; they were 0.88, 0.92, and 0.83 respectively after the first extirpation day and 25.0, 28.50, and 21.25 respectively after the second extirpation day, which showed an decrease at first, and then increase at the second day. To the flora that in feces, on the contrast, only the richness showed the same tendency. Escherichia coli and Pseudomonas were the dominant flora both in vagina and feces, Rhodococcus presented in specific both in vagina and feces, while Staphylococcus and Aeromonas hydrophila presented specific only in vagina, and Acinetobacter were specific in feces. All the results indicated that uterus extirpation exerted a great impact on microbial community structure in vagina, Escherichia coli and Pseudomonas in vagina might be derived from feces.