In cardiac myocytes, regulation of mitochondrial Ca2+ is important for cellular signaling and cardiac contraction. Ca2+ entry into the mitochondria is mediated by a highly selective Ca2+ channel called the mitochondrial calcium uniporter, which consists of a pore-forming subunit MCU and regulatory subunits such as MICU1. Although pharmacological regulation of the mitochondrial Ca2+ influx is a promising approach to controlling the cellular functions, a cell-permeable and specific inhibitor of the mitochondrial calcium uniporter has not yet been developed. Here, we identify a novel cell-permeable inhibitor of the uniporter by a high-throughput screening of 120 000 small-molecule compounds. In our study, DS16570511 dose-dependently inhibited serum-induced mitochondrial Ca2+ influx in HEK293A cells with an IC50 of 7 ?M. DS16570511 inhibited Ca2+ uptake of isolated mitochondria from human cells, rat heart and pig heart. Overexpression of hMCU or hMICU1 in HEK293A cells increased mitochondrial Ca2+ influx, and the increases were completely suppressed by the pretreatment with DS16570511. DS16570511 also blocks mitochondrial Ca2+ overload in a Langendorff perfused beating rat heart. Interestingly, DS16570511 increased cardiac contractility without affecting heart rate in the perfused heart. These results show that DS16570511 is a novel cell-permeable inhibitor of the mitochondrial calcium uniporter and applicable for control of the cardiac functions.