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Soybean agglutinin (SBA) is a heat-sensitive anti-nutritional factor (ANF). It affects nutrient absorption and causes organism poisoning. This study explored the SBA passivation ability and mechanism by ultra-high pressure (HHP), a non-thermal food processing technology. The results indicated that more than 500 MPa HHP treatment reduced the SBA activity by destroying its secondary and tertiary structures. Also, the cell and animal experiments showed that HHP treatment reduced the cytotoxicity of SBA, improved the mice’s body weight, and alleviated liver, kidney, and digestive tract damage in Vivo. These results demonstrated that HHP had a high passivation efficiency against the SBA, thereby HHP promoting the safety of soybean products. This study provided supporting evidence for ultra-high-pressure treatment applications in soybean processing.