Multi-drug-resistant Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis were isolated from the tissue samples of a giant panda. The E. coli strain SH-YH-DH that we isolated carried a self-transferable plasmid associated with multi-drug-resistant genes ( blaCTX-M-55, blaTEM-1, sul1, floR, strB, aac(6')-Ib, and tetA/R). This strain exhibited phenotypic resistance to gentamicin, cefotaxime, ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and tetracycline. We report the participation of multi-drug-resistant E. coli in a extraintestinal polyinfection in giant pandas and that further research is warranted to pay attention to the threat of multi-drug-resistant bacteria in the panda population.