In July 2021, a disease with a high mortality rate broke out in freshwater cultured hybrid sturgeon in Zhengzhou, Henan Province. A dominant strain, H-701, was isolated from diseased fish; physiological changes in diseased fish were investigated and molecular identification, biochemical characterization, and pathogenicity and drug sensitivity tests of H-701 were performed. The 16S rRNA gene sequence of H-701 was 99.86% homologous with that of Vibrio metschnikovii in GenBank. The 50% lethal dose of H-701 was 3.72 ± 0.929 × 104 CFU/g fish weight. The proportion of monocytes, neutrophils, and eosinophils in the blood of diseased sturgeon increased significantly, whereas the proportion of lymphocytes decreased. In diseased fish, the serum levels of total protein, albumin, globulin, and alkaline phosphatase decreased significantly, and those of aspartate aminotransferase, alanine aminotransferase, and complement C3 increased significantly. There were obvious pathological changes in several tissues of the diseased fish. H-701 was sensitive to antibiotics such as florfenicol, enrofloxacin, and doxycycline. This study not only demonstrated that V. metschnikovii was the cause of death of a large number of hybrid sturgeon but also revealed its potential risk in hybrid sturgeon aquaculture. The results provide a basis for the diagnosis and prevention of this disease.