The mitochondrial calcium uniporter (MCU) complex mediates Ca(2+) entry into mitochondria, which plays a crucial role in regulating cellular energy metabolism and apoptosis. Dysregulation of MCU is implicated in various diseases, such as neurodegenerative disorders, cardiac diseases, and cancer. Despite its importance, developing specific and clinically viable MCU inhibitors is challenging. Here, Berberine, a well-established drug with a documented safety profile, is identified as a potent MCU inhibitor through a virtual screening of an FDA-approved drug library. Berberine localizes within mitochondria and directly binds to the juxtamembrane loop domain of MCU. This binding disrupts the interaction of MCU with its essential regulator, EMRE, thereby inhibiting rapid Ca(2+) entry into the mitochondria. Notably, Berberine pretreatment reduces mitochondrial Ca(2+) overload and mitigates ischemia/reperfusion-induced myocardial injury in mice. These findings establish Berberine as a potent MCU inhibitor, offering a safe therapeutic strategy for diseases associated with dysregulated mitochondrial calcium homeostasis.