Background: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca ²⁺ ( _m Ca ²⁺ ) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. Methods: We created a stable MCUB knockout cell line ( MCUB ^−/− ) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca ²⁺ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. Results: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2–dependent cooperative activation of the mtCU, thereby decreasing _m Ca ²⁺ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit _m Ca ²⁺ overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in _m Ca ²⁺ uptake impair mitochondrial energetics and contractile function. Conclusions: We detail a new regulatory mechanism to modulate mtCU function and _m Ca ²⁺ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate _m Ca ²⁺ uptake and cellular physiology.