In August 2019, an outbreak of mass mortality occurred in the Pacific oysters farmed in Shandong, China, resulting in over 60% mortality. Bacterial pathogens were isolated from moribund oysters, among which one strain identified as gram-negative rod-shaped bacterium, named as Cg5, was further characterized in this study. Based on 16S rRNA phylogenetic analysis and biochemical characterization, Cg5 was determined to be Vibrio alginolyticus. Injection of V. alginolyticus isolate into healthy oysters revealed its pathogenicity, which showed same symptoms and histopathological changes as the naturally infected oysters. Analysis of growth capability of the isolate at different temperatures showed that high temperature was conducive to pathogen proliferation. These results implied that high temperature could facilitate the growth and pathogenicity of V. alginolyticus, suggesting the inducement of disease outbreak by high temperature in summer. Additionally, the important virulence genes, including AspA, Collagenase, flaB, fur, ompW, tlh and toxR, were all detected from the V. alginolyticus, further supporting its strong pathogenicity. To the best of our knowledge, this is the first report of V. alginolyticus as a causative pathogen associated with mass summer mortality of the Pacific oysters cultured in China, which provides valuable information for disease surveillance and genetic breeding program to produce oyster strains with high disease resistance.