Introduction: The transport of the universal second messenger Ca2+ across the mitochondrial inner membrane plays a key role in numerous physiological processes such as cell cycle control, signal transduction and cell death pathways. The mitochondrial Ca2+ uptake machinery consists of several components amongst others the channel forming mitochondrial Ca2+ uniporter (MCU), its regulatory components including the dominant-negative pore-forming subunit MCUb, the essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 (MICU1). MICU1 acts as gatekeeper for MCU; under low Ca2+ conditions it forms hexamers that inhibit MCU activity while at high Ca2+ concentrations it disassembles to dimmers that do not further prevent MCU activity. Methods: In this study a Förster resonance energy transfer (FRET)-based live-cell approach was applied to dynamically monitor the arrangement of MICU1 multimers after Ca2+ mobilization by an IP3-generating agonist. For this purpose MICU1 fused to either cyan fluorescent protein (MICU1-CFP) or yellow fluorescent protein (MICU1-YFP) were overexpressed in HeLa cells to follow the (dis-)assembly of MICU1 multimers by dynamic FRET imaging. Results: Cytosolic Ca2+ elevation triggers disassembly of MICU1 hexamers with an EC50 of 4.4 µM. MICU1 disassembly precedes the mitochondrial Ca2+ uptake. These results indicate that the transfer of cytosolic Ca2+ into mitochondria consists of four steps: (1) elevation of cytosolic/intermembrane Ca2+, (2) Ca2+ binding to MICU1, (3) MICU1 disassembly and (4) MCU activation and Ca2+ transport into the matrix. The rearrangement of MICU1 is independent of mitochondrial membrane potential and expression levels of MCU/EMRE. Conclusion: This study revealed further insights into the regulation of mitochondrial Ca2+ uptake as well as the regulation and the (re-)organization of the gatekeeping component MICU1. The second messenger Ca2+ was shown to be the main regulator of the (dis-)assembly of MICU1 oligomers prior to mitochondrial Ca2+ uptake. Our approach allowed correlation of the kinetics of MICU1 (re-)organization with cellular Ca2+ dynamics and revealed that the rearrangement of MICU1 multimers strictly correlates with the cytosolic Ca2+ concentration and is rapidly reversible.