<p>The mitochondrial Ca²⁺ uniporter mediates mitochondrial Ca²⁺ uptake to regulate cellular bioenergetics, Ca²⁺ signaling and survival, but excessive activity triggers Ca²⁺ overload and tissue injury. Cells counter this threat by expressing MCUb, a paralog of the uniporter’s pore-forming MCU subunit, to attenuate uniporter activity. Despite harboring the conserved Ca²⁺-coordinating DIME motif, MCUb paradoxically lacks conductance, a defining yet enigmatic feature underlying its uniporter-inhibitory role. Here, we demonstrate that MCUb’s non-conductivity stems from its inability to bind EMRE, a subunit essential for uniporter function, and that its N-terminal domain (NTD) exerts autoinhibition. Reinstating EMRE binding and relieving NTD-mediated inhibition rebuild Ca²⁺ conductance in MCUb, reaching ~80% of MCU activity. Wild-type MCUb exhibits ~30% of the inhibitory capacity of a pore-disrupting E249A variant, indicating that MCUb is a modest, rather than potent, negative regulator. These findings reveal how MCU-MCUb paralog divergence endows the uniporter with regulatory plasticity to fine-tune mitochondrial Ca²⁺ homeostasis.</p>