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The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases. Synopsis: Mitochondrial calcium uptake through the uniporter channel MCUC is critical to cell signaling, metabolism, physiology, and disease. This study provides an unbiased and quantitative map of the MCUC interactome and its remodelling, both under resting conditions and after genetic perturbations. Tandem affinity purification/mass-spectrometry-based approach identifies the MCUC protein interaction network. Mitochondrial inner membrane protein EFHD1 interacts with MCUC and inhibits mitochondrial calcium uptake. Loss of MCUB subunit results in an expansion and greater interconnection of the MCU protein network. A dynamic map of the MCUC interaction landscape reveals inhibition of mitochondrial calcium uptake by the mitochondrial intermembrane space protein EFHD1. [ABSTRACT FROM AUTHOR]