<p>In most eukaryotes, the mitochondrial calcium uniporter (MCU) mediates Ca²⁺ influx into the mitochondrial matrix through a process regulated by MICUs and the EMRE. In <i>Dictyostelium discoideum</i>, a model organism for amoebozoans that lack an EMRE, the MCU complex consists solely of the MCU and MICU. Most likely, therefore, the mechanism by which <i>Dd</i>MICU regulates the <i>Dd</i>MCU differs from the extensively studied metazoan MCU-EMRE-MICU system. Here, we report the crystal structure of Ca²⁺-bound <i>Dd</i>MICU at 2.5 Å resolution. Unlike human MICUs, which contain two Ca²⁺-binding EF-hand motifs, <i>Dd</i>MICU possesses three EF-hand motifs, each with a submicromolar Ca²⁺ binding affinity. The overall structure of <i>Dd</i>MICU is comparable to that of human MICUs, and their well-conserved dimer interface interactions are similar. In addition to the face-to-face dimer observed in human MICUs, <i>Dd</i>MICU forms a head-to-head dimer with multimeric states that equilibrate between tetrameric and dimeric forms, depending on the solution ionic strength. Moreover, the C-helix of <i>Dd</i>MICU plays a critical role in membrane binding. These findings provide a molecular basis for the unique mechanism regulating Ca²⁺ uptake by MICUs in an EMRE-free system and offer insight into the evolution and functional diversity of the MCU complex in non-metazoan organisms.</p>