Abstract:[Background] Hafnia alvei, a Gram-negative bacillus, an opportunistic pathogen, a saprophytic bacterium, is commonly detected in human and animal intestines, sewage, soil, and dairy products and can cause septicemia in human and animal, showing the potential to result in diarrhoea. [Objective] To isolate, identify and analyze the biological characteristics of potential pathogenic bacteria in the death of a barking deer (Muntjac) in Kunming Jiaozi Snow Mountain Nature Reserve. [Methods] A part of the intestinal tissue of the dead barking deer was collected aseptically for bacterial isolation and identification, and the isolate was subjected to drug sensitivity test and animal regression test. [Results] The isolate was identified as Hafnia alvei and named KMJZXS0312. It was resistant to 7 antibiotics including penicillin and cefthiophene, intermediately resistant to florfenicol, kanamycin, furazolidone, and amoxicillin, and sensitive to 13 antibiotics such as enrofloxacin and compound sulfamethoxazole. The animal regression test showed that the strain caused flatulence in the stomach and intestinal tract, thin and translucent intestine, spot hemorrhage in the liver, pinpoint hemorrhage in the lung. In addition, the strain caused degeneration and swelling of hepatocytes, as well as loose and lightly staining of the cytoplasm. Finally, it led to the death of mice.[Conclusion] A pathogenic H. alvei strain was isolated from a barking deer intestinal tissue. The biological characterization and drug sensitivity test in this study provide new biological information of the strain, which has important public health significance.