Abstract: Non-Saccharomyces cerevisiae often appears on the surface of berries in the early natural fermentation of fruit wine, particularly on the most common varieties of Kloeckera apiculata and Hanseniaspora uvarum. However, the growth, reproduction, and metabolism of the non-Saccharomyces cerevisiae can easily produce ethanol and other undesired components, thereby causing the quality changes in most fruit and vegetable products, generally regarded the phenomenon as a spoilage strain. Thermal treatment is a common way to kill spoilage strains in fruit and vegetable juice, fresh fruit and non-alcoholic beverages. This approach can effectively inhibit the growth of spoilage strains. However, the loss of heat-sensitive active substances is accompanied during the treatment, leading inevitably to the decline in sensory quality of food. Alternatively, non-thermal sterilization technology, represented by high hydrostatic pressure (HHP), has widely been applied in food processing, which can effectively overcome the disadvantages of thermal treatment. Most previous studies were relatively comprehensive focuses on the bacteria in the sterilization mechanism of HHP on food microorganisms, but it is still lacking on the non-Saccharomyces cerevisiae. Furthermore, the growth and reproduction of non-Saccharomyces cerevisiae are the main factors resulting in the microbial spoilage of berry products. Therefore, it is necessary to effectively inhibit the physiological activity of non-Saccharomyces cerevisiae, thereby improving the product quality, while reducing the loss of flavor value. One kind of non-Saccharomyces cerevisiae, the Hanseniaspora opuntiae can be separated from natural berries. Therefore, it is feasible to investigate the damage mechanism of Hanseniaspora opuntiae with different sterilization treatments. In this study, Hanseniaspora opuntiae was isolated from naturally putrefied Patriot blueberry (Semen trigonellae) juice, and then treated with HHP to explore the damage mechanism, where the pressure of 300 MPa and the duration of 300 s were selected as the experimental condition. A flow cytometry, circular dichroism, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were adopted to determine the effects of HHP on the cell membrane, cell morphology and intracellular substances. The experimental results showed that the death count of HHP was 4.36 lg (CFU/mL), indicating an excellent inactivation effect. After treatment with HHP, the cell membrane was destroyed, and 70.50% of Hanseniaspora opuntiae was stained by PI. Nucleic acid, protein and ions were leaked out, where the OD260nm and OD280nm reached 1.132 and 0.374, respectively, and the electrical conductivity rose to 26 μS/cm, indicating that the membrane permeability of most cells increased. Meanwhile, the activities of Na+/K+-ATPase, Ca2+/Mg2+-ATPase, and total ATPase decreased, where the inhibition rates reached 31.13%, 16.01%, and 20.06%, respectively. The results of AO staining and circular dichroism spectrum demonstrated that the structure of nucleic acid and protein were changed, proving that the HHP can affect intracellular substances. In the images of SEM and TEM, there were perforations on the cell membrane and outflow of cytoplasm in large quantities. The results indicated that the HHP damage to Hanseniaspora opuntiae was due mainly to the destruction of the cell membrane, which caused the intracellular substances to lose their activities in an HHP treatment.