BACKGROUND: Oxidative stress is a significant cause in the occurrence of cardiac insufficiency. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase2 (NOX2)-derived reactive oxygen species (ROS) play a pivotal role in oxidative stress-induced excitation-contraction decoupling. Stachydrine hydrochloride (Sta) reduces pressure overload-induced cardiac insufficiency, which may be related to the NOX2-ROS pathway, as demonstrated by our earlier research. However, the mechanism through which Sta specifically affects NOX2 remains unknown. PURPOSE: In order to investigate whether Sta plays a cardioprotective role by inhibiting NOX2 activity, we explored the specific mechanism by which Sta improves cardiac function by affecting NOX2-mediated oxidative stress in this study. METHODS: Molecular docking and cellular thermal shift assay (CETSA) were performed to verify whether Sta can bind to individual subunits of NOX2. We induced models of cardiac insufficiency in the compensatory phase (cardiac hypertrophy) by phenylephrine (PE) in vivo and in vitro and treated with Sta and GSK2795039 (NOX2 inhibitor). Cardiac function and structure were observed by echocardiography analysis. We detected the expression and localization of NOX2 subunits and calcium channel proteins, also detected the activities of ROS and NOX2, SOD, and GSH, and observed intracardiac calcium homeostasis and systolic-diastolic function in cardiomyocytes. Secondly, we used adenovirus and adeno-associated virus transfection for cardiac-specific overexpression of NOX2 in vivo and in vitro respectively, and also treated with Sta to observe NOX2 activation indexes and ROS levels, cardiac function and cardiomyocyte function in mice. RESULTS: Prior to our investigation, we discovered that Sta could bind to NOX2 through molecular docking and CETSA. The findings demonstrated that Sta decreased the expression levels of gp91 CONCLUSIONS: Our study innovatively verified the key role of NOX2 in cardiac insufficiency. Sta downgrades NOX2's activity by suppressing the protein level of gp91