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Cardiovascular diseases (CVDs) are the leading causes of premature death and disability in people around the world. Therefore, the prevention and treatment of CVDs has become an important subject. In this study, we verified the thrombolytic activities of a nattokinase‐like protease named NK‐01 in vivo. Label‐free liquid chromatography–tandem mass spectrometry (LC‐MS/MS) technique was used in our study. NK‐01 could inhibit the activity of coagulation factors though the up‐regulation of proteinase C inhibitors and protein S. NK‐01 also could inhibit the angiotensinogen conversion to AngII and promote the degradation of kininogen to reduce the blood pressure. In addition, NK‐01 could increase the content of paraoxonase 1, which could prevent atherosclerosis. In our study, we found that NK‐01 cloud effect some key proteins which participant in CVDs associated metabolic processes such as coagulation system, blood pressure, and atherosclerosis. Taken together, the underlying molecular mechanisms for the biological beneficial of NK‐01 were investigated. Our proteomic study will provide further theoretical basis for application of NK in prevention or adjuvant treatment in biomedicine areas. [ABSTRACT FROM AUTHOR]