Abstract: In order to confirm a suspected case of Haemophilus Parasuis infection in a pig, in this experiment, bacteria were isolated form the collected infected tissue, and the isolated strains were identified by morphology observation, culture characterization, biochemical characterization and PCR methods. Meanwhile, agar diffusion test, genotyping, sequencing analysis and pathogenicity test were carried out on the isolated strain. The results showed that the isolated strains were gram-negative bacteria with pleomorphism, and the colony morphology was round, smooth, gray-white, semi-transparent with neat edge. It showed NAD-dependent, typical satellite growth phenomenon, and no hemolysis phenomenon. The biochemical test results were consistent with the biochemical characteristics of Haemophilus Parasuis. A 1 090 bp band was amplified by PCR, and was identified as serovar 5 by agar gel diffusion test. The isolate was identified by genotyping and only 468 bp fragment was amplified, which was confirmed to be serovar 5 by sequencing comparison. In the pathogenicity test of pig, there was no abnormality in the blank control pigs, and the infected pigs showed typical clinical symptoms of fever, arthritis and fibrinous polyserositis on the surface of multiple organs of chest and abdomen caused by Haemophilus parasuis. The results suggested that the infected pig was infected with Haemophilus parasuis, and the serovar 5 Haemophilus parasuis strain was isolated in this research.